U.S. patent application number 17/248907 was filed with the patent office on 2021-09-02 for jak inhibitors containing a 4-membered heterocyclic amide.
This patent application is currently assigned to THERAVANCE BIOPHARMA R&D IP, LLC. The applicant listed for this patent is THERAVANCE BIOPHARMA R&D IP, LLC. Invention is credited to GARY E.L. BRANDT, PAUL R. FATHEREE, CAMERON SMITH, STEVEN D.E. SULLIVAN, LORI JEAN VAN ORDEN.
Application Number | 20210269436 17/248907 |
Document ID | / |
Family ID | 1000005594982 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210269436 |
Kind Code |
A1 |
FATHEREE; PAUL R. ; et
al. |
September 2, 2021 |
JAK INHIBITORS CONTAINING A 4-MEMBERED HETEROCYCLIC AMIDE
Abstract
The invention provides compounds of formula (I): ##STR00001##
which contain a 4-membered heterocyclic amide, where the variables
are defined in the specification, or a pharmaceutically-acceptable
salt thereof, that are useful as JAK kinase inhibitors. The
invention also provides pharmaceutical compositions comprising such
compounds, methods of using such compounds to treat respiratory
diseases, and processes and intermediates useful for preparing such
compounds.
Inventors: |
FATHEREE; PAUL R.; (SAN
FRANCISCO, CA) ; BRANDT; GARY E.L.; (ALAMEDA, CA)
; SMITH; CAMERON; (SAN BRUNO, CA) ; SULLIVAN;
STEVEN D.E.; (SAN FRANCISCO, CA) ; VAN ORDEN; LORI
JEAN; (SAN FRANCISCO, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THERAVANCE BIOPHARMA R&D IP, LLC |
SOUTH SAN FRANCISCO |
CA |
US |
|
|
Assignee: |
THERAVANCE BIOPHARMA R&D IP,
LLC
SOUTH SAN FRANCISCO
CA
|
Family ID: |
1000005594982 |
Appl. No.: |
17/248907 |
Filed: |
February 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16701426 |
Dec 3, 2019 |
10954237 |
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17248907 |
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16511410 |
Jul 15, 2019 |
10550118 |
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16701426 |
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16222647 |
Dec 17, 2018 |
10392386 |
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16511410 |
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15915418 |
Mar 8, 2018 |
10196393 |
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16222647 |
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62469073 |
Mar 9, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 11/00 20180101; C07D 471/04 20130101; C07D 519/00
20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; C07D 519/00 20060101 C07D519/00; A61P 11/06 20060101
A61P011/06; A61P 11/00 20060101 A61P011/00 |
Claims
1-21. (canceled)
22. A method of inhibiting a Janus kinase in a mammal comprising
administering to the mammal a compound of formula (I): ##STR00034##
wherein: R.sup.1 is selected from hydrogen, C.sub.1-3alkyl, and
C.sub.3-6cycloalkyl, and X is --C(O)R.sup.2 wherein R.sup.2 is
--NR.sup.13R.sup.14, wherein R.sup.13 and R.sup.14 are taken
together with the nitrogen atom to which they are attached form a
4-membered heterocyclyl, wherein the heterocyclyl is optionally
substituted with --NR.sup.5R.sup.6 and R.sup.7, R.sup.5 and R.sup.6
are independently C.sub.1-3alkyl or R.sup.5 and R.sup.6 taken
together with the nitrogen atom to which they are attached form a
5- or 6-membered heterocyclyl optionally including an oxygen atom,
R.sup.7 is C.sub.1-3alkyl, optionally substituted with a 5- or
6-membered heterocyclyl containing one nitrogen atom, or a
pharmaceutically-acceptable salt thereof.
23. The method of claim 22, wherein R.sup.1 is hydrogen or
C.sub.1-3alkyl.
24. The method of claim 22, wherein R.sup.13 and R.sup.14 taken
together with the nitrogen atom to which they are attached form a
4-membered heterocyclyl, wherein the heterocyclyl is optionally
substituted with --NR.sup.5R.sup.6 and R.sup.7, or R.sup.5 and
R.sup.6 are independently C.sub.1-3alkyl or R.sup.5 and R.sup.6
taken together with the nitrogen atom to which they are attached
form a 5- or 6-membered heterocyclyl, and R.sup.7 is
C.sub.1-3alkyl, optionally substituted with pyrrolidinyl.
25. The method of claim 22, wherein the compound has the formula
(II): ##STR00035## wherein: R.sup.1 is C.sub.1-3alkyl; R.sup.2 is
##STR00036## wherein R.sup.5 and R.sup.6 are independently
C.sub.1-3alkyl or R.sup.5 and R.sup.6 taken together form
--(CH.sub.2).sub.4-5--, R.sup.7 is hydrogen or C.sub.1-3alkyl, or a
pharmaceutically-acceptable salt thereof.
26. The method of claim 25, wherein R.sup.5 and R.sup.6 are
C.sub.1-3alkyl.
27. The method of claim 22, wherein the compound has the formula:
##STR00037## or a pharmaceutically-acceptable salt thereof.
28. The method of claim 22, wherein the compound has the formula:
##STR00038##
29. The method of claim 22, wherein the compound, or a
pharmaceutically-acceptable salt thereof, inhibits JAK1, JAK2,
JAK3, and TYK2.
30. The method of claim 27, wherein the compound, or a
pharmaceutically-acceptable salt thereof, inhibits JAK1, JAK2,
JAK3, and TYK2.
31. The method of claim 27, wherein the mammal is a human.
32. The method of claim 31, wherein the compound, or a
pharmaceutically-acceptable salt thereof, is administered in a
pharmaceutical composition further comprising a
pharmaceutically-acceptable carrier.
33. The method of claim 32, wherein the pharmaceutical composition
is administered to the lungs by inhalation.
34. The method of claim 33, wherein the pharmaceutical composition
is administered by nebulized administration.
35. The method of claim 33, wherein the pharmaceutical composition
is administered with an inhaler delivery device.
36. The method of claim 35, wherein the inhaler delivery device is
selected from the group consisting of a dry powder inhaler, a
metered-dose inhaler, and a nebulizer inhaler.
37. The method of claim 33, wherein the pharmaceutical composition
is in the form of a solution.
38. The method of claim 33, wherein the pharmaceutical composition
is in the form of a dry powder.
39. The method of claim 32, wherein the pharmaceutical composition
contains from 0.01 to 95% by weight of the compound.
40. The method of claim 32, wherein the pharmaceutical composition
contains from 0.05 to 30% by weight of the compound.
41. The method of claim 32, wherein the pharmaceutical composition
contains from 0.1% to 10% by weight of the compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/469,073, filed on Mar. 9, 2017, the disclosure
of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The invention is directed to compounds useful as JAK kinase
inhibitors. The invention is also directed to pharmaceutical
compositions comprising such compounds, methods of using such
compounds to treat respiratory diseases, and processes and
intermediates useful for preparing such compounds.
State of the Art
[0003] Asthma is a chronic disease of the airways for which there
are no preventions or cures. The disease is characterized by
inflammation, fibrosis, hyper-responsiveness, and remodeling of the
airways, all of which contribute to airflow limitation. An
estimated 300 million people worldwide suffer from asthma and it is
estimated that the number of people with asthma will grow by more
than 100 million by 2025. In the United States, asthma afflicts
about 6% to 8% of the population, making it one of the most common
chronic diseases in the country. Although most patients can achieve
control of asthma symptoms with the use of inhaled corticosteroids
that may be combined with a leukotriene modifier and/or a long
acting beta agonist, there remains a subset of patients with severe
asthma whose disease is not controlled by conventional therapies.
Severe persistent asthma is defined as disease that remains
uncontrolled on high doses of inhaled corticosteroids. While severe
asthmatics are estimated to account for approximately 5% of all
asthma sufferers, they have a high risk of morbidity and mortality
and are responsible for a disproportionate share of health care
resource utilization among asthmatics. There remains a need for
novel therapies to treat these patients.
[0004] Cytokines are intercellular signaling molecules which
include chemokines, interferons, interleukins, lymphokines, and
tumor necrosis factor. Cytokines are critical for normal cell
growth and immunoregulation but also drive immune-mediated diseases
and contribute to the growth of malignant cells. Elevated levels of
many cytokines have been implicated in the pathology of asthma
inflammation. For example, antibody-based therapies targeted at
interleukins (IL)-5, and 13 have been shown to provide clinical
benefit in subsets of severe asthma patients. Among the cytokines
implicated in asthma inflammation, many act through signaling
pathways dependent upon the Janus family of tyrosine kinases
(JAKs), which signal through the Signal Transducer and Activator of
Transcription (STAT) family of transcription factors. Cytokines
implicated in asthma inflammation which signal through the JAK-STAT
pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13,
IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP),
interferon-.gamma. (IFN.gamma.) and granulocyte-macrophage
colony-stimulating factor (GM-CSF).
[0005] The JAK family comprises four members, JAK1, JAK2, JAK3, and
tyrosine kinase 2 (TYK2). Binding of cytokine to a JAK-dependent
cytokine receptor induces receptor dimerization which results in
phosphorylation of tyrosine residues on the JAK kinase, effecting
JAK activation. Phosphorylated JAKs, in turn, bind and
phosphorylate various STAT proteins which dimerize, internalize in
the cell nucleus and directly modulate gene transcription, leading,
among other effects, to the downstream effects associated with
inflammatory disease. The JAKs usually associate with cytokine
receptors in pairs as homodimers or heterodimers. Specific
cytokines are associated with specific JAK pairings. Each of the
four members of the JAK family is implicated in the signaling of at
least one of the cytokines associated with asthma inflammation.
Consequently, a chemical inhibitor with pan-activity against all
members of the JAK family could modulate a broad range of
pro-inflammatory pathways that contribute to severe asthma.
[0006] However, the broad anti-inflammatory effect of such
inhibitors could suppress normal immune cell function, potentially
leading to increased risk of infection. Evidence of increased
infection risk has been observed with the JAK inhibitor
tofacitinib, which is dosed orally for the treatment of rheumatoid
arthritis. In asthma, inflammation is localized to the respiratory
tract. Inflammation of the airways is characteristic of other
respiratory diseases in addition to asthma. Chronic obstructive
pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,
interstitial lung diseases (including idiopathic pulmonary
fibrosis), acute lung injury, acute respiratory distress syndrome,
bronchitis, emphysema, bronchiolitis obliterans, and sarcoidosis
are also respiratory tract diseases in which the pathophysiology is
believed to be related to JAK-signaling cytokines. Local
administration of a JAK inhibitor to the lungs by inhalation offers
the potential to be therapeutically efficacious by delivering a
potent anti-cytokine agent directly to the site of action, limiting
systemic exposure and therefore limiting the potential for adverse
systemic immunosuppression. The need remains for a potent JAK
inhibitor suitable for local administration to the lungs for
treatment of respiratory disease.
[0007] JAK-signaling cytokines also play a major role in the
activation of T cells, a sub-type of immune cells that is central
to many immune processes. Pathological T cell activation is
critical in the etiology of multiple respiratory diseases.
Autoreactive T cells play a role in bronchiolitis obliterans
organizing pneumonia (also termed COS). Similar to COS the etiology
of lung transplant rejections is linked to an aberrant T cell
activation of the recipients T cells by the transplanted donor
lung. Lung transplant rejections may occur early as Primary Graft
Dysfunction (PGD), organizing pneumonia (OP), acute rejection (AR)
or lymphocytic bronchiolitis (LB) or they may occur years after
lung transplantation as Chronic Lung Allograft Dysfunction (CLAD).
CLAD was previously known as bronchiolitis obliterans (BO) but now
is considered a syndrome that can have different pathological
manifestations including BO, restrictive CLAD (rCLAD or RAS) and
neutrophilic allograft dysfunction. Chronic lung allograft
dysfunction (CLAD) is a major challenge in long-term management of
lung transplant recipients as it causes a transplanted lung to
progressively lose functionality (Gauthier et al., Curr Transplant
Rep., 2016, 3(3), 185-191). CLAD is poorly responsive to treatment
and therefore, there remains a need for effective compounds capable
of preventing or treating this condition. Several JAK-dependent
cytokines such as IFN.gamma. and IL-5 are up-regulated in CLAD and
lung transplant rejection (Berastegui et al, Clin Transplant. 2017,
31, e12898). Moreover, high lung levels of CXCR3 chemokines such as
CXCL9 and CXCL10 which are downstream of JAK-dependent IFN
signaling, are linked to worse outcomes in lung transplant patients
(Shino et al, PLOS One, 2017, 12 (7), e0180281). Systemic JAK
inhibition has been shown to be effective in kidney transplant
rejection (Vicenti et al., American Journal of Transplantation,
2012, 12, 2446-56). Therefore, JAK inhibitors have the potential to
be effective in treating or preventing lung transplant rejection
and CLAD. Similar T cell activation events as described as the
basis for lung transplant rejection also are considered the main
driver of lung graft-versus-host disease (GVHD) which can occur
post hematopoietic stem cell transplants. Similar to CLAD, lung
GVHD is a chronic progressive condition with extremely poor
outcomes and no treatments are currently approved. A retrospective,
multicenter survey study of 95 patients with steroid-refractory
acute or chronic GVHD who received the systemic JAK inhibitor
ruxolitinib as salvage therapy demonstrated complete or partial
response to ruxolitinib in the majority of patients including those
with lung GVHD (Zeiser et al, Leukemia, 2015, 29, 10, 2062-68). As
systemic JAK inhibition is associated with serious adverse events
and a small therapeutic index, the need remains for an inhaled
lung-directed, non-systemic JAK inhibitor to prevent and/or treat
lung transplant rejection or lung GVHD.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides novel compounds having
activity as JAK kinase inhibitors.
[0009] Accordingly, the invention provides a compound of formula
(I):
##STR00002##
wherein:
[0010] R.sup.1 is selected from hydrogen, C.sub.1-3alkyl, and
C.sub.3-6cycloalkyl, and X is --C(O)R.sup.2
[0011] wherein [0012] R.sup.2 is --NR.sup.13R.sup.14, wherein
[0013] R.sup.13 and R.sup.14 taken together with the nitrogen atom
to which they are attached form a 4-membered heterocyclyl, wherein
the heterocyclyl is optionally substituted with --NR.sup.5R.sup.6
and R.sup.7, [0014] R.sup.5 and R.sup.6 are independently
C.sub.1-3alkyl or R.sup.5 and R.sup.6 taken together with the
nitrogen atom to which they are attached form a 5- or 6-membered
heterocyclyl optionally including an oxygen atom, [0015] R.sup.7 is
C.sub.1-3alkyl, optionally substituted with a 5- or 6-membered
heterocyclyl containing one nitrogen atom, [0016] or a
pharmaceutically-acceptable salt thereof.
[0017] As used hereinafter, the phrase "compound of formula (I)"
means a compound of formula (I) or a pharmaceutically acceptable
salt thereof; i.e., this phrase means a compound of formula (I) in
free base form or in a pharmaceutically acceptable salt form unless
otherwise indicated.
[0018] The invention also provides a pharmaceutical composition
comprising a compound of the invention and a
pharmaceutically-acceptable carrier.
[0019] The invention also provides a method of treating respiratory
disease, in particular, asthma, in a mammal, the method comprising
administering to the mammal a therapeutically effective amount of a
compound or of a pharmaceutical composition of the invention. In
separate and distinct aspects, the invention also provides
synthetic processes and intermediates described herein, which are
useful for preparing compounds of the invention.
[0020] The invention also provides a compound of the invention as
described herein for use in medical therapy, as well as the use of
a compound of the invention in the manufacture of a formulation or
medicament for treating respiratory disease in a mammal.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Among other aspects, the invention provides JAK kinase
inhibitors of formula (I), pharmaceutically-acceptable salts
thereof, and intermediates for the preparation thereof. The
following substituents and values are intended to provide
representative examples of various aspects of this invention. These
representative values are intended to further define such aspects
and are not intended to exclude other values or limit the scope of
the invention.
[0022] In a specific aspect, R.sup.1 is selected from hydrogen,
C.sub.1-3alkyl, and C.sub.3-6cycloalkyl.
[0023] In another specific aspect, R.sup.1 is selected from
hydrogen and C.sub.1-3alkyl. In yet another specific aspect,
R.sup.1 is C.sub.1-3alkyl.
[0024] Specific values of R.sup.1 include, but are not limited to,
methyl, ethyl, n-propyl, and isopropyl.
[0025] In a specific aspect, R.sup.1 is selected from hydrogen and
C.sub.1-3alkyl and X is --C(O)R.sup.2 wherein R.sup.2 is
##STR00003##
wherein R.sup.5 and R.sup.6 are independently C.sub.1-3alkyl or
R.sup.5 and R.sup.6 taken together form --(CH.sub.2).sub.4-5-- and
R.sup.7 is hydrogen or C.sub.1-3alkyl.
[0026] In another specific aspect, R.sup.1 is selected from
hydrogen and C.sub.1-3alkyl and X is --C(O)R.sup.2 wherein R.sup.2
is
##STR00004##
wherein R.sup.5 and R.sup.6 are both methyl or R.sup.5 and R.sup.6
taken together form --(CH.sub.2).sub.5--; and R.sup.7 is hydrogen
or methyl.
[0027] In another aspect, the invention provides a compound of
formula (II):
##STR00005##
wherein:
[0028] R.sup.1 is C.sub.1-3alkyl;
[0029] R.sup.2 is
##STR00006##
[0030] wherein [0031] R.sup.5 and R.sup.6 are independently
C.sub.1-3alkyl or R.sup.5 and R.sup.6 taken together form
[0032] --(CH.sub.2).sub.4-5--, R.sup.7 is hydrogen or
C.sub.1-3alkyl,
or a pharmaceutically-acceptable salt thereof.
[0033] In another specific aspect, R.sup.1 is C.sub.1-3alkyl;
[0034] R.sup.2 is
##STR00007##
[0035] wherein
[0036] R.sup.5 and R.sup.6 are both methyl or R.sup.5 and R.sup.6
taken together form
[0037] --(CH.sub.2).sub.5--, and R.sup.7 is hydrogen or methyl.
[0038] In yet another aspect, the invention provides a compound
wherein the compound is
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-flu-
oro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]-
pyridin-6-yl)methanone, or a pharmaceutically-acceptable salt
thereof.
[0039] In yet another aspect, the invention provides a compound
wherein the compound is
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-flu-
oro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]-
pyridin-6-yl)methanone.
[0040] In yet another aspect, the invention provides a compound
wherein the compound is
(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hyd-
roxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-
-yl)methanone, or a pharmaceutically-acceptable salt thereof.
[0041] In yet another aspect, the invention provides a compound
wherein the compound is
(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hyd-
roxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-
-yl)methanone.
[0042] In yet another aspect, the invention provides a compound
selected from the following compounds:
[0043]
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydro-
xyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c-
]pyridin-6-yl)methanone,
[0044]
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydro-
xyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]py-
ridin-6-yl)methanone,
[0045]
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-fluor-
o-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo-
[4,5-c]pyridin-6-yl)methanone,
[0046] and pharmaceutically-acceptable salts thereof.
[0047] In yet another aspect, the invention provides a compound
selected from the following compounds:
[0048]
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydro-
xyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c-
]pyridin-6-yl)methanone,
[0049]
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydro-
xyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]py-
ridin-6-yl)methanone, and
[0050]
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-fluor-
o-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo-
[4,5-c]pyridin-6-yl)methanone.
[0051] In yet another aspect, the invention provides
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphen-
yl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-
tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone of the
formula
##STR00008##
or a pharmaceutically-acceptable salt thereof.
[0052] In yet another aspect, the invention provides a compound of
the formula
##STR00009##
[0053] In one aspect, the invention provides the compounds of
Examples 2, 4, 8, and Table 1 below.
[0054] Chemical structures are named herein according to IUPAC
conventions as implemented in ChemDraw software (PerkinElmer, Inc.,
Cambridge, Mass.). For example, the compound:
##STR00010##
[0055] is designated as
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphen-
yl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyrid-
in-6-yl)methanone.
[0056] Furthermore, the imidazo portion of the
tetrahydroimidazopyridine moiety in the structure of formula (I)
exists in tautomeric forms, illustrated below for a fragment of the
compound of Example 1
##STR00011##
According to the IUPAC convention, these representations give rise
to different numbering of the atoms of the imidazole portion:
2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine
(structure A) vs.
2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine
(structure B). It will be understood that although structures are
shown, or named, in a particular form, the invention also includes
the tautomer thereof The compounds of the invention may contain one
or more chiral centers and therefore, such compounds (and
intermediates thereof) can exist as racemic mixtures; pure
stereoisomers (i.e., enantiomers or diastereomers);
stereoisomer-enriched mixtures and the like. Chiral compounds shown
or named herein without a defined stereochemistry at a chiral
center are intended to include any or all possible stereoisomer
variations at the undefined stereocenter unless otherwise
indicated. The depiction or naming of a particular stereoisomer
means the indicated stereocenter has the designated stereochemistry
with the understanding that minor amounts of other stereoisomers
may also be present unless otherwise indicated, provided that the
utility of the depicted or named compound is not eliminated by the
presence of another stereoisomer. Compounds of formula (I) also
contain several basic groups (e.g., amino groups) and therefore,
such compounds can exist as the free base or in various salt forms,
such a mono-protonated salt form, a di-protonated salt form, a
tri-protonated salt form, or mixtures thereof. All such forms are
included within the scope of this invention, unless otherwise
indicated.
[0057] This invention also includes isotopically-labeled compounds
of formula (I), i.e., compounds of formula (I) where one or more
atom has been replaced or enriched with an atom having the same
atomic number but an atomic mass different from the atomic mass
that predominates in nature. Examples of isotopes that may be
incorporated into a compound of formula (I) include, but are not
limited to, .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C,
.sup.13N, .sup.15N, .sup.15O, .sup.17O, and .sup.18O. Of particular
interest are compounds of formula (I) enriched in tritium or
carbon-14, which compounds can be used, for example, in tissue
distribution studies. Also of particular interest are compounds of
formula (I) enriched in deuterium especially at a site of
metabolism, which compounds are expected to have greater metabolic
stability. Additionally of particular interest are compounds of
formula (I) enriched in a positron emitting isotope, such as
.sup.11C, .sup.15O and .sup.13N, which compounds can be used, for
example, in Positron Emission Tomography (PET) studies.
[0058] Definitions
[0059] When describing this invention including its various aspects
and embodiments, the following terms have the following meanings,
unless otherwise indicated.
[0060] The term "alkyl" means a monovalent saturated hydrocarbon
group which may be linear or branched or combinations thereof.
Unless otherwise defined, such alkyl groups typically contain from
1 to 10 carbon atoms. Representative alkyl groups include, by way
of example, methyl (Me), ethyl (Et), n-propyl (n-Pr) or (nPr),
isopropyl (i-Pr) or (iPr), n-butyl (n-Bu) or (nBu), sec-butyl,
isobutyl, tert-butyl (t-Bu) or (tBu), n-pentyl, n-hexyl,
2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl, 2-ethylbutyl,
2,2-dimethylpentyl, 2-propylpentyl, and the like.
[0061] When a specific number of carbon atoms are intended for a
particular term, the number of carbon atoms is shown preceding the
term. For example, the term "C.sub.1-3 alkyl" means an alkyl group
having from 1 to 3 carbon atoms wherein the carbon atoms are in any
chemically-acceptable configuration, including linear or branched
configurations.
[0062] The term "cycloalkyl" means a monovalent saturated
carbocyclic group which may be monocyclic or multicyclic. Unless
otherwise defined, such cycloalkyl groups typically contain from 3
to 10 carbon atoms. Representative cycloalkyl groups include, by
way of example, cyclopropyl (cPr), cyclobutyl (cBu), cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like.
[0063] The term "heterocyclyl", "heterocycle", "heterocyclic", or
"heterocyclic ring" means a monovalent saturated or partially
unsaturated cyclic non-aromatic group, having from 3 to 10 total
ring atoms, wherein the ring contains from 2 to 9 carbon ring atoms
and from 1 to 4 ring heteroatoms selected from nitrogen, oxygen,
and sulfur. Heterocyclic groups may be monocyclic or multicyclic
(i.e., fused or bridged). Representative heterocyclyl groups
include, by way of example, pyrrolidinyl, piperidinyl, piperazinyl,
imidazolidinyl, morpholinyl, thiomorpholyl, indolin-3-yl,
2-imidazolinyl, tetrahydropyranyl,
1,2,3,4-tetrahydroisoquinolin-2-yl, quinuclidinyl,
7-azanorbornanyl, nortropanyl, and the like, where the point of
attachment is at any available carbon or nitrogen ring atom. Where
the context makes the point of attachment of the heterocyclic group
evident, such groups may alternatively be referred to as a
non-valent species, i.e. pyrrolidine, piperidine, piperazine,
imidazole, tetrahydropyran etc.
[0064] The term "halo" means fluoro, chloro, bromo or iodo.
[0065] The term "therapeutically effective amount" means an amount
sufficient to effect treatment when administered to a patient in
need of treatment.
[0066] The term "treating" or "treatment" means preventing,
ameliorating or suppressing the medical condition, disease or
disorder being treated (e.g., a respiratory disease) in a patient
(particularly a human); or alleviating the symptoms of the medical
condition, disease or disorder.
[0067] The term "pharmaceutically acceptable salt" means a salt
that is acceptable for administration to a patient or a mammal,
such as a human (e.g., salts having acceptable mammalian safety for
a given dosage regime). Representative pharmaceutically acceptable
salts include salts of acetic, ascorbic, benzenesulfonic, benzoic,
camphorsulfonic, citric, ethanesulfonic, edisylic, fumaric,
gentisic, gluconic, glucoronic, glutamic, hippuric, hydrobromic,
hydrochloric, isethionic, lactic, lactobionic, maleic, malic,
mandelic, methanesulfonic, mucic, naphthalenesulfonic,
naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,
nitric, orotic, pamoic, pantothenic, phosphoric, succinic,
sulfuric, tartaric, p-toluenesulfonic and xinafoic acid, and the
like.
[0068] The term "salt thereof" means a compound formed when the
hydrogen of an acid is replaced by a cation, such as a metal cation
or an organic cation and the like. For example, the cation can be a
protonated form of a compound of formula (I), i.e. a form where one
or more amino groups have been protonated by an acid. Typically,
the salt is a pharmaceutically acceptable salt, although this is
not required for salts of intermediate compounds that are not
intended for administration to a patient.
[0069] The term "amino-protecting group" means a protecting group
suitable for preventing undesired reactions at an amino nitrogen.
Representative amino-protecting groups include, but are not limited
to, formyl; acyl groups, for example alkanoyl groups, such as
acetyl and tri-fluoroacetyl; alkoxycarbonyl groups, such as tent
butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as
benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);
arylmethyl groups, such as benzyl (Bn), trityl (Tr), and
1,1-di-(4'-methoxyphenyl)methyl; silyl groups, such as
trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),
[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like.
[0070] The term "hydroxy-protecting group" means a protecting group
suitable for preventing undesired reactions at a hydroxy group.
Representative hydroxy-protecting groups include, but are not
limited to, alkyl groups, such as methyl, ethyl, and tent-butyl;
acyl groups, for example alkanoyl groups, such as acetyl;
arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB),
9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); silyl
groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl
(TBS); and the like.
[0071] Numerous protecting groups, and their introduction and
removal, are described in T. W. Greene and P. G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York
General Synthetic Procedures
[0072] Compounds of this invention, and intermediates thereof, can
be prepared according to the following general methods and
procedures using commercially-available or routinely-prepared
starting materials and reagents. The substituents and variables
(e.g., R.sup.1, R.sup.2, etc.) used in the following schemes have
the same meanings as those defined elsewhere herein unless
otherwise indicated. Additionally, compounds having an acidic or
basic atom or functional group may be used or may be produced as a
salt unless otherwise indicated (in some cases, the use of a salt
in a particular reaction will require conversion of the salt to a
non-salt form, e.g., a free base, using routine procedures before
conducting the reaction).
[0073] Although a particular embodiment of the present invention
may be shown or described in the following procedures, those
skilled in the art will recognize that other embodiments or aspects
of the present invention can also be prepared using such procedures
or by using other methods, reagents, and starting materials know to
those skilled in the art. In particular, it will be appreciated
that compounds of the invention may be prepared by a variety of
process routes in which reactants are combined in different orders
to provide different intermediates en route to producing final
products.
[0074] A general method of preparing final compounds of the
invention in which the variable X is defined as --C(O)R.sup.2 and
R.sup.1 is C.sub.1-3alkyl utilizes a key intermediate 1 and an
amine of formula 2 as illustrated generally in Scheme 1 and, in
particular, for an example in which R.sup.2 is defined as
##STR00012##
to specifically exemplify a representative amide final product of
formula (II).
##STR00013##
To prepare amide compounds of formula (II), the carboxylic acid of
formula 1 is reacted with amine 2 according to typical amide bond
formation conditions. Typically, carboxylic acid 1 is contacted
with between about 1 and about 4 equivalents of amine 2 in the
presence of an excess of base. As shown in the examples below, the
amide bond formation reaction may utilize coupling agents, such as
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (HATU) or other amide coupling agents known in
the art. The reaction is typically conducted at room temperature
for between about 2 and about 24 hours or until the reaction is
substantially complete.
[0075] The carboxylic acid of formula 1 may be prepared as
illustrated in Scheme 2
##STR00014## ##STR00015##
where Pg.sup.1 represents a hydroxy-protecting group and Pg.sup.2,
Pg.sup.3, and Pg.sup.4 represent different amino-protecting groups.
As described in the examples below, a useful choice of protecting
groups is benzyl or methyl as Pg.sup.1, tetrahydropyranyl (THP) as
Pg.sup.2, tert-butoxycarbonyl (Boc) or benzyl as Pg.sup.3, and
[2-(trimethylsilyl)ethoxy]methyl (SEM) as Pg.sup.4. The first step
of Scheme 2 is the palladium catalyzed Stille coupling of
intermediate 3 with intermediate 4 where the phenyl-indazole
intermediate 3 has the trimethylstannyl moiety and the reaction
partner 4 is iodine substituted. The reaction is typically
conducted at elevated temperature, for example, at between about
80.degree. C. and about 180.degree. C. for between about 10 and
about 24 hours or until the reaction is substantially complete.
[0076] When benzyl is used as Pg.sup.1, in the next step, the
methyl ester of intermediate 5 is converted to a benzyl ester in
intermediate 6 by reaction of 5 with benzyl alcohol. Both benzyl
protecting groups are conveniently removed by palladium catalyzed
hydrogenation to provide intermediate 7 which may be fully
deprotected by reaction with acid, typically hydrochloric acid. In
a final step, the substituent R.sup.1 is added by reductive
alkylation of intermediate 8 with a reagent R.sup.1a where R.sup.1a
is an aldehyde or ketone defined such that upon reduction, R.sup.1
is produced. For example, to add a methyl substituent R.sup.1,
formaldehyde is used as reagent R.sup.1a, to add an isopropyl
moiety as substituent R.sup.1, acetone is used as reagent R.sup.1a.
The reaction is typically conducted in the presence of a reducing
agent such as sodium cyanoborohydride or sodium
triacetoxyborohydride or the like at ambient temperature for a
period of about 10 to about 24 hours or until the reaction is
substantially complete.
[0077] Intermediates 3 and 4 may be prepared from commercial or
easily prepared starting materials, as described in detail below.
In particular, a process for preparing intermediate 3 in which
Pg.sup.1 is benzyl and Pg.sup.2 is THP uses the Suzuki-Miyaura
coupling of compound 9 with compound 10 followed by conventional
reactions to add the trimethylstannyl group.
##STR00016##
[0078] Intermediate 4 may be prepared from compound 11, which is
commercially available in racemic and stereospecific forms and may
also be prepared from histidine. Accordingly, in a method aspect,
the invention provides a process of preparing a compound of formula
(II) or a pharmaceutically acceptable salt thereof, the process
comprising reacting a compound of formula 1 with a compound of
formula 2, as illustrated in Scheme 1 to provide a compound of
formula (II) or a pharmaceutically acceptable salt thereof.
[0079] In a further method aspect, the invention provides a process
of preparing a compound of formula 1, the process comprising
reacting a compound of formula 8 with R.sup.1a in the presence of a
reducing agent, wherein R.sup.1a is an aldehyde or ketone defined
such that upon reductive alkylation the substituent R.sup.1,
wherein R.sup.1 is C.sub.1-3alkyl, is attached to the compound of
formula 8 to provide the compound of formula 1.
[0080] In an additional method aspect, the invention provides a
process of preparing a compound of formula 8 the process comprising
deprotecting a compound of formula 7.
[0081] In yet another aspect, the invention provides a compound of
formula 1 and compounds of formula 7 and 8, useful in preparing a
compound of formula 1.
Pharmaceutical Compositions
[0082] The compounds of the invention and
pharmaceutically-acceptable salts thereof are typically used in the
form of a pharmaceutical composition or formulation. Such
pharmaceutical compositions may advantageously be administered to a
patient by inhalation. In addition, pharmaceutical compositions may
be administered by any acceptable route of administration
including, but not limited to, oral, rectal, nasal, topical
(including transdermal) and parenteral modes of administration.
[0083] Accordingly, in one of its compositions aspects, the
invention is directed to a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier or excipient and a compound of
formula (I), where, as defined above, "compound of formula (I)"
means a compound of formula (I) or a pharmaceutically-acceptable
salt thereof. Optionally, such pharmaceutical compositions may
contain other therapeutic and/or formulating agents if desired.
When discussing compositions and uses thereof, the "compound of the
invention" may also be referred to herein as the "active agent". As
used herein, the term "compound of the invention" is intended to
include all compounds encompassed by formula (I) as well as the
species embodied in formula (II) and pharmaceutically-acceptable
salts thereof.
[0084] The pharmaceutical compositions of the invention typically
contain a therapeutically effective amount of a compound of the
present invention. Those skilled in the art will recognize,
however, that a pharmaceutical composition may contain more than a
therapeutically effective amount, i.e., bulk compositions, or less
than a therapeutically effective amount, i.e., individual unit
doses designed for multiple administration to achieve a
therapeutically effective amount.
[0085] Typically, such pharmaceutical compositions will contain
from about 0.01 to about 95% by weight of the active agent;
including, for example, from about 0.05 to about 30% by weight; and
from about 0.1% to about 10% by weight of the active agent.
[0086] Any conventional carrier or excipient may be used in the
pharmaceutical compositions of the invention. The choice of a
particular carrier or excipient, or combinations of carriers or
excipients, will depend on the mode of administration being used to
treat a particular patient or type of medical condition or disease
state. In this regard, the preparation of a suitable pharmaceutical
composition for a particular mode of administration is well within
the scope of those skilled in the pharmaceutical arts.
Additionally, the carriers or excipients used in the pharmaceutical
compositions of this invention are commercially-available. By way
of further illustration, conventional formulation techniques are
described in Remington: The Science and Practice of Pharmacy, 20th
Edition, Lippincott Williams & White, Baltimore, Md. (2000);
and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug
Delivery Systems, 7th Edition, Lippincott Williams & White,
Baltimore, Md. (1999).
[0087] Representative examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, the following: sugars, such as lactose, glucose and sucrose;
starches, such as corn starch and potato starch; cellulose, such as
microcrystalline cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical compositions.
[0088] Pharmaceutical compositions are typically prepared by
thoroughly and intimately mixing or blending the active agent with
a pharmaceutically-acceptable carrier and one or more optional
ingredients. The resulting uniformly blended mixture can then be
shaped or loaded into tablets, capsules, pills and the like using
conventional procedures and equipment.
[0089] In one aspect, the pharmaceutical composition is suitable
for inhaled administration. Pharmaceutical compositions for inhaled
administration are typically in the form of an aerosol or a powder.
Such compositions are generally administered using inhaler delivery
devices, such as a dry powder inhaler (DPI), a metered-dose inhaler
(MDI), a nebulizer inhaler, or a similar delivery device.
[0090] In a particular embodiment, the pharmaceutical composition
is administered by inhalation using a dry powder inhaler. Such dry
powder inhalers typically administer the pharmaceutical composition
as a free-flowing powder that is dispersed in a patient's
air-stream during inspiration. In order to achieve a free-flowing
powder composition, the therapeutic agent is typically formulated
with a suitable excipient such as lactose, starch, mannitol,
dextrose, polylactic acid (PLA), polylactide-co-glycolide (PLGA) or
combinations thereof. Typically, the therapeutic agent is
micronized and combined with a suitable carrier to form a
composition suitable for inhalation.
[0091] A representative pharmaceutical composition for use in a dry
powder inhaler comprises lactose and a compound of the invention in
micronized form. Such a dry powder composition can be made, for
example, by combining dry milled lactose with the therapeutic agent
and then dry blending the components. The composition is then
typically loaded into a dry powder dispenser, or into inhalation
cartridges or capsules for use with a dry powder delivery
device.
[0092] Dry powder inhaler delivery devices suitable for
administering therapeutic agents by inhalation are described in the
art and examples of such devices are commercially available. For
example, representative dry powder inhaler delivery devices or
products include Aeolizer (Novartis); Airmax (IVAX); ClickHaler
(Innovata Biomed); Diskhaler (GlaxoSmithKline); Diskus/Accuhaler
(GlaxoSmithKline); Ellipta (GlaxoSmithKline); Easyhaler (Orion
Pharma); Eclipse (Aventis); FlowCaps (Hovione); Handihaler
(Boehringer Ingelheim); Pulvinal (Chiesi); Rotahaler
(GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma); Twisthaler
(Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler (Aventis);
and the like.
[0093] In another particular embodiment, the pharmaceutical
composition is administered by inhalation using a metered-dose
inhaler. Such metered-dose inhalers typically discharge a measured
amount of a therapeutic agent using a compressed propellant gas.
Accordingly, pharmaceutical compositions administered using a
metered-dose inhaler typically comprise a solution or suspension of
the therapeutic agent in a liquefied propellant. Any suitable
liquefied propellant may be employed including hydrofluoroalkanes
(HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and
1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and
chlorofluorocarbons, such as CCl.sub.3F. In a particular
embodiment, the propellant is hydrofluoroalkanes. In some
embodiments, the hydrofluoroalkane formulation contains a
co-solvent, such as ethanol or pentane, and/or a surfactant, such
as sorbitan trioleate, oleic acid, lecithin, and glycerin.
[0094] A representative pharmaceutical composition for use in a
metered-dose inhaler comprises from about 0.01% to about 5% by
weight of a compound of the invention; from about 0% to about 20%
by weight ethanol; and from about 0% to about 5% by weight
surfactant; with the remainder being an HFA propellant. Such
compositions are typically prepared by adding chilled or
pressurized hydrofluoroalkane to a suitable container containing
the therapeutic agent, ethanol (if present) and the surfactant (if
present). To prepare a suspension, the therapeutic agent is
micronized and then combined with the propellant. The composition
is then loaded into an aerosol canister, which typically forms a
portion of a metered-dose inhaler device.
[0095] Metered-dose inhaler devices suitable for administering
therapeutic agents by inhalation are described in the art and
examples of such devices are commercially available. For example,
representative metered-dose inhaler devices or products include
[0096] AeroBid Inhaler System (Forest Pharmaceuticals); Atrovent
Inhalation Aerosol (Boehringer Ingelheim); Flovent
(GlaxoSmithKline); Maxair Inhaler (3M); Proventil Inhaler
(Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); and the
like.
[0097] In another particular aspect, the pharmaceutical composition
is administered by inhalation using a nebulizer inhaler. Such
nebulizer devices typically produce a stream of high velocity air
that causes the pharmaceutical composition to spray as a mist that
is carried into the patient's respiratory tract. Accordingly, when
formulated for use in a nebulizer inhaler, the therapeutic agent
can be dissolved in a suitable carrier to form a solution.
Alternatively, the therapeutic agent can be micronized or
nanomilled and combined with a suitable carrier to form a
suspension.
[0098] A representative pharmaceutical composition for use in a
nebulizer inhaler comprises a solution or suspension comprising
from about 0.05 .mu.g/mL to about 20 mg/mL of a compound of the
invention and excipients compatible with nebulized formulations. In
one embodiment, the solution has a pH of about 3 to about 8.
Nebulizer devices suitable for administering therapeutic agents by
inhalation are described in the art and examples of such devices
are commercially available. For example, representative nebulizer
devices or products include the Respimat Softmist Inhalaler
(Boehringer Ingelheim); the AERx Pulmonary Delivery System (Aradigm
Corp.); the PARI LC Plus Reusable Nebulizer (Pari GmbH); and the
like.
[0099] In yet another aspect, the pharmaceutical compositions of
the invention may alternatively be prepared in a dosage form
intended for oral administration. Suitable pharmaceutical
compositions for oral administration may be in the form of
capsules, tablets, pills, lozenges, cachets, dragees, powders,
granules; or as a solution or a suspension in an aqueous or
non-aqueous liquid; or as an oil-in-water or water-in-oil liquid
emulsion; or as an elixir or syrup; and the like; each containing a
predetermined amount of a compound of the present invention as an
active ingredient.
[0100] When intended for oral administration in a solid dosage
form, the pharmaceutical compositions of the invention will
typically comprise the active agent and one or more
pharmaceutically-acceptable carriers, such as sodium citrate or
dicalcium phosphate. Optionally or alternatively, such solid dosage
forms may also comprise: fillers or extenders, binders, humectants,
solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, coloring agents, and buffering agents.
Release agents, wetting agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the pharmaceutical compositions of the
invention.
[0101] Alternative formulations may also include controlled release
formulations, liquid dosage forms for oral administration,
transdermal patches, and parenteral formulations. Conventional
excipients and methods of preparation of such alternative
formulations are described, for example, in the reference by
Remington, supra.
[0102] The following non-limiting examples illustrate
representative pharmaceutical compositions of the present
invention.
[0103] Dry Powder Composition
[0104] A micronized compound of formula (I) (1 g) is blended with
milled lactose (25 g). This blended mixture is then loaded into
individual blisters of a peelable blister pack in an amount
sufficient to provide between about 0.1 mg to about 4 mg of the
compound of formula I per dose. The contents of the blisters are
administered using a dry powder inhaler.
[0105] Dry Powder Composition
[0106] A micronized compound of formula (I) (1 g) is blended with
milled lactose (20 g) to form a bulk composition having a weight
ratio of compound to milled lactose of 1:20.
[0107] The blended composition is packed into a dry powder
inhalation device capable of delivering between about 0.1 mg to
about 4 mg of the compound of formula I per dose.
[0108] Metered-Dose Inhaler Composition
[0109] A micronized compound of formula (I) (10 g) is dispersed in
a solution prepared by dissolving lecithin (0.2 g) in demineralized
water (200 mL). The resulting suspension is spray dried and then
micronized to form a micronized composition comprising particles
having a mean diameter less than about 1.5 .mu.m. The micronized
composition is then loaded into metered-dose inhaler cartridges
containing pressurized 1,1,1,2-tetrafluoroethane in an amount
sufficient to provide about 0.1 mg to about 4 mg of the compound of
formula I per dose when administered by the metered dose
inhaler.
[0110] Nebulizer Composition
[0111] A compound of formula (I) (25 mg) is dissolved in a solution
containing 1.5-2.5 equivalents of hydrochloric acid, followed by
addition of sodium hydroxide to adjust the pH to 3.5 to 5.5 and 3%
by weight of glycerol. The solution is stirred well until all the
components are dissolved. The solution is administered using a
nebulizer device that provides about 0.1 mg to about 4 mg of the
compound of formula I per dose.
Utility
[0112] The JAK inhibitors of the invention have been designed for
the treatment of inflammatory and fibrotic disease of the
respiratory tract. In particular, the compounds have been designed
to enable delivery of a potent anti-cytokine agent directly to the
site of action of respiratory disease in the lung while limiting
systemic exposure.
[0113] The compounds of the invention have been shown to be potent
inhibitors of the JAK family of enzymes: JAK1, JAK2, JAK3, and
TYK2. In addition, the compounds have demonstrated potent
inhibition of pro-inflammatory and pro-fibrotic cytokines without
exhibiting cytotoxicity in cellular assays. It has been recognized
that the broad anti-inflammatory effect of JAK inhibitors could
suppress normal immune cell function, potentially leading to
increased risk of infection. The present compounds have therefore
been optimized to limit absorption from the lung into the plasma,
thus minimizing the risk of immunosuppression.
[0114] As described in the experimental section below, the
absorption and distribution of typical compounds has been profiled
in preclinical assays. Selected compounds tested in mice showed, at
the same time, high concentration in lung tissue and low absorption
into plasma. Compounds tested in mouse exhibited exposure in lung
from one to two orders of magnitude greater than exposure in
plasma. The compounds also exhibited significant retention in the
mouse lung as evidenced by a lung half-life greater than about 5
hours. Importantly, the concentration of test compound in the mouse
lung has been shown to correlate with a predicted pharmacodynamic
effect of JAK enzyme inhibition. Compounds of the invention have
been shown to inhibit an effect of the pro-inflammatory cytokine
IL-13 in mouse lung tissue. Specifically, the compounds have
demonstrated dose and concentration dependent inhibition of
IL-13-induced phosphorylation of STAT6 in lung tissue which
provides evidence of local lung JAK target engagement in vivo. This
effect has been observed when the pro-inflammatory cytokine IL-13
is administered 4 hours after administration of the test compound,
providing further evidence of significant retention in the
lung.
[0115] Tested compounds have been demonstrated to exhibit both
potent inhibitory activity at the cellular level and significant
retention in lung tissue. Extensive investigation by the present
inventors has determined that while it is possible to identify
compounds that are potent at the cellular level or compounds that
show significant retention in the lung, it is far more difficult to
discover compounds that exhibit both desirable characteristics at
the same time.
[0116] The anti-inflammatory activity of JAK inhibitors has been
robustly demonstrated in preclinical models of asthma (Malaviya et
al., Int Immunopharmacol, 2010, 10, 829,-836; Matsunaga et al.,
Biochem and Biophys Res Commun, 2011, 404, 261-267; Kudlacz et al.,
Eur J Pharmacol, 2008, 582, 154-161). Accordingly, the compounds of
the invention are expected to be useful for the treatment of
inflammatory respiratory disorders, in particular, asthma.
Inflammation and fibrosis of the lung is characteristic of other
respiratory diseases in addition to asthma such as chronic
obstructive pulmonary disease (COPD), cystic fibrosis (CF),
pneumonitis, interstitial lung diseases (including idiopathic
pulmonary fibrosis), acute lung injury, acute respiratory distress
syndrome, bronchitis, emphysema, bronchiolitis obliterans, and
sarcoidosis. The present compounds, therefore, are also expected to
be useful for the treatment of chronic obstructive pulmonary
disease, cystic fibrosis, pneumonitis, interstitial lung diseases
(including idiopathic pulmonary fibrosis), acute lung injury, acute
respiratory distress syndrome, bronchitis, emphysema, bronchiolitis
obliterans, and sarcoidosis.
[0117] The compounds of the disclosure have demonstrated inhibition
of human T cell activation, inhibition of cytokines associated with
inflammation, and activity on human eosinophils and in rodent lung
eosinophilia models. Therefore, the compounds of the disclosure are
likely to be useful for the treatment of certain specific
respiratory diseases. Eosinophilic airway inflammation is a
characteristic feature of diseases collectively termed eosinophilic
lung diseases (Cottin et al., Clin. Chest. Med., 2016, 37(3),
535-56). Eosinophilic diseases have been associated with IL-4,
IL-13 and IL-5 signaling. Eosinophilic lung diseases include
infections (especially helminthic infections), drug-induced
pneumonitis (induced for example by therapeutic drugs such as
antibiotics, phenytoin, or 1-tryptophan), fungal-induced
pneumonitis (e.g. allergic bronchopulmonary aspergillosis),
hypersensitivity pneumonitis and eosinophilic granulomatosis with
polyangiitis (formerly known as Churg-Strauss syndrome).
Eosinophilic lung diseases of unknown etiology include idiopathic
acute eosinophilic pneumoni, idiopathic chronic eosinophilic
pneumonia, hypereosinophilic syndrome, and Loffler syndrome. The
compounds of the disclosure have been shown to significantly reduce
lung eosinophilia in the rodent airway model and to potently
inhibit IL-13, IL-4, and IL-2 signaling in cellular assays. In
addition, the compound of example 2 has been demonstrated to
potently inhibit IL-5 mediated human eosinophil survival.
[0118] A polymorphism in the IL-6 gene has been associated with
elevated IL-6 levels and an increased risk of developing pulmonary
arterial hypertension (PAH) (Fang et al., J Am Soc Hypertens.,
2017, 11(3), 171-177). Corroborating the role of IL-6 in PAH,
inhibition of the IL-6 receptor chain gp130 ameliorated the disease
in a rat model of PAH (Huang et al., Can J Cardiol., 2016, 32(11),
1356.e1-1356.e10). The compound of example 2 has been shown to
inhibit IL-6 signaling.
[0119] Cytokines such as IFN.gamma., IL-12 and IL-6 have been
implicated in a range of non-allergic lung diseases such as
sarcoidosis, and lymphangioleiomyomatosis (El-Hashemite et al., Am.
J. Respir. Cell Mol. Biol., 2005, 33, 227-230, and El-Hashemite et
al., Cancer Res., 2004, 64, 3436-3443). The compound of example 2
has also been shown to inhibit IL-6 and IFN.gamma. signaling.
[0120] Bronchiectasis and infiltrative pulmonary diseases are
diseases associated with chronic neutrophilic inflammation. The
compound of example 2 has been shown to inhibit cytokines that are
associated with neutrophilic inflammation (e.g. IL-6,
IFN.gamma.).
[0121] Pathological T cell activation is critical in the etiology
of multiple respiratory diseases. Autoreactive T cells play a role
in bronchiolitis obliterans organizing pneumonia (also termed COS).
Similar to COS the etiology of lung transplant rejections is linked
to an aberrant T cell activation of the recipients T cells by the
transplanted donor lung. Lung transplant rejections may occur early
as Primary Graft Dysfunction (PGD), organizing pneumonia (OP),
acute rejection (AR) or lymphocytic bronchiolitis (LB) or they may
occur years after lung transplantation as Chronic Lung Allograft
Dysfunction (CLAD). CLAD was previously known as bronchiolitis
obliterans (BO) but now is considered a syndrome that can have
different pathological manifestations including BO, restrictive
CLAD (rCLAD or RAS) and neutrophilic allograft dysfunction. Chronic
lung allograft dysfunction (CLAD) is a major challenge in long-term
management of lung transplant recipients as it causes a
transplanted lung to progressively lose functionality (Gauthier et
al., Curr Transplant Rep., 2016, 3(3), 185-191). CLAD is poorly
responsive to treatment and therefore, there remains a need for
effective compounds capable of preventing or treating this
condition. Several JAK-dependent cytokines such as IFN.gamma. and
IL-5 are up-regulated in CLAD and lung transplant rejection
(Berastegui et al, Clin Transplant. 2017, 31, e12898). Moreover,
high lung levels of CXCR3 chemokines such as CXCL9 and CXCL10 which
are downstream of JAK-dependent IFN signaling, are linked to worse
outcomes in lung transplant patients (Shino et al, PLOS One, 2017,
12 (7), e0180281). Systemic JAK inhibition has been shown to be
effective in kidney transplant rejection (Vicenti et al., American
Journal of Transplantation, 2012, 12, 2446-56). Therefore, JAK
inhibitors have the potential to be effective in treating or
preventing lung transplant rejection and CLAD. Similar T cell
activation events as described as the basis for lung transplant
rejection also are considered the main driver of lung
graft-versus-host disease (GVHD) which can occur post hematopoietic
stem cell transplants. Similar to CLAD, lung GVHD is a chronic
progressive condition with extremely poor outcomes and no
treatments are currently approved. A retrospective, multicenter
survey study of 95 patients with steroid-refractory acute or
chronic GVHD who received the systemic JAK inhibitor ruxolitinib as
salvage therapy demonstrated complete or partial response to
ruxolitinib in the majority of patients including those with lung
GVHD (Zeiser et al, Leukemia, 2015, 29, 10, 2062-68). As systemic
JAK inhibition is associated with serious adverse events and a
small therapeutic index, the need remains for an inhaled
lung-directed, non-systemic JAK inhibitor to prevent and/or treat
lung transplant rejection or lung GVHD. The compounds of the
disclosure have the characteristics required to meet this need.
More recently, immune-checkpoint inhibitor induced pneumonitis,
another T cell mediated lung disease emerged with the increased use
of immune-checkpoint inhibitors. In cancer patients treated with
these T cell stimulating agents, fatal pneumonitis can develop. The
compound of example 2 has been shown to inhibit the anti-CD3 and
IL-2 induced release of IFN.gamma. from activated human peripheral
blood-isolated T cells and the production of CXCL9 and CXCL10 in
airway epithelial cells and thus has the potential to present a
novel treatment for these underserved serious respiratory
diseases.
[0122] In one aspect, therefore, the invention provides a method of
treating a respiratory disease in a mammal (e.g., a human), the
method comprising administering to the mammal a
therapeutically-effective amount of a compound of the invention or
of a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a compound of the
invention.
[0123] In one aspect, the respiratory disease is asthma, chronic
obstructive pulmonary disease, cystic fibrosis, pneumonitis,
chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF),
pneumonitis, interstitial lung diseases (including idiopathic
pulmonary fibrosis), acute lung injury, acute respiratory distress
syndrome, bronchitis, emphysema, bronchiolitis obliterans, or
sarcoidosis. In another aspect, the respiratory disease is asthma
or chronic obstructive pulmonary disease.
[0124] In one aspect, the respiratory disease is a lung infection,
an eosinophilic disease, a helminthic infection, pulmonary arterial
hypertension, sarcoidosis, lymphangioleiomyomatosis,
bronchiectasis, an infiltrative pulmonary disease, drug-induced
pneumonitis, fungal induced pneumonitis, allergic bronchopulmonary
aspergillosis, hypersensitivity pneumonitis, eosinophilic
granulomatosis with polyangiitis, idiopathic acute eosinophilic
pneumonia, idiopathic chronic eosinophilic pneumonia,
hypereosinophilic syndrome, Loffler syndrome, bronchiolitis
obliterans organizing pneumonia, acute and chronic lung transplant
rejections (including PGD, OP, LB, AR and CLAD, BO, restrictive
CLAD and neutrophilic allograft dysfunction), lung
graft-versus-host disease bronchiolitis obliterans organizing
pneumonia, pulmonary arterial hypertension, bronchiectasis, or
immune-checkpoint-inhibitor induced pneumonitis.
[0125] The invention further provides a method of treating asthma
in a mammal, the method comprising administering to the mammal a
therapeutically-effective amount of a compound of the invention or
of a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a compound of the
invention.
[0126] When used to treat asthma, the compounds of the invention
will typically be administered in a single daily dose or in
multiple doses per day, although other forms of administration may
be used. The amount of active agent administered per dose or the
total amount administered per day will typically be determined by a
physician, in the light of the relevant circumstances, including
the condition to be treated, the chosen route of administration,
the actual compound administered and its relative activity, the
age, weight, and response of the individual patient, the severity
of the patient's symptoms, and the like.
[0127] The invention further provides a method of treating a
respiratory disease (including but not limited to the disease
described herein) in a mammal, the method comprising administering
to the mammal a therapeutically-effective amount of a compound of
the invention or of a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a compound of the
invention.
[0128] When used to treat a respiratory disease (including but not
limited to the disease described herein), the compounds of the
invention will typically be administered in a single daily dose or
in multiple doses per day, although other forms of administration
may be used. The amount of active agent administered per dose or
the total amount administered per day will typically be determined
by a physician, in the light of the relevant circumstances,
including the condition to be treated, the chosen route of
administration, the actual compound administered and its relative
activity, the age, weight, and response of the individual patient,
the severity of the patient's symptoms, and the like.
[0129] As JAK inhibitors, the compounds of the disclosure may also
be useful for a variety of other diseases. The compounds of the
disclosure may be useful for a variety of gastrointestinal
inflammatory indications that include, but are not limited to,
inflammatory bowel disease, ulcerative colitis (proctosigmoiditis,
pancolitis, ulcerative proctitis and left-sided colitis), Crohn's
disease, collagenous colitis, lymphocytic colitis,
[0130] Behcet's disease, celiac disease, immune checkpoint
inhibitor induced colitis, ileitis, eosinophilic esophagitis, graft
versus host disease-related colitis, and infectious colitis.
Ulcerative colitis (Reimund et al., J Clin Immunology, 1996, 16,
144-150), Crohn's disease (Woywodt et al., Eur J Gastroenterology
Hepatology, 1999, 11, 267-276), collagenous colitis (Kumawat et
al., Mol Immunology, 2013, 55, 355-364), lymphocytic colitis
(Kumawat et al., 2013), eosinophilic esophagitis
(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graft
versus host disease-related colitis (Coghill et al., Blood, 2001,
117, 3268-3276), infectious colitis (Stallmach et al., Int J
Colorectal Dis, 2004, 19, 308-315), Behcet's disease (Zhou et al.,
Autoimmun Rev, 2012, 11, 699-704), celiac disease (de Nitto et al.,
World J Gastroenterol, 2009, 15, 4609-4614), immune checkpoint
inhibitor induced colitis (e.g., CTLA-4 inhibitor-induced colitis;
(Yano et al., J Translation Med, 2014, 12, 191), PD-1- or
PD-L1-inhibitor-induced colitis), and ileitis (Yamamoto et al., Dig
Liver Dis, 2008, 40, 253-259) are characterized by elevation of
certain pro-inflammatory cytokine levels. As many pro-inflammatory
cytokines signal via JAK activation, compounds described in this
application may be able to alleviate the inflammation and provide
symptom relief. In particular, the compounds of the disclosure may
be useful for the induction and maintenance of remission of
ulcerative colitis, and for the treatment of Crohn's disease,
immune checkpoint inhibitor induced colitis, and the
gastrointestinal adverse effects in graft versus host disease. In
one aspect, therefore, the invention provides a method of treating
a gastrointestinal inflammatory disease in a mammal (e.g., a
human), the method comprising administering to the mammal a
compound of the disclosure or a pharmaceutically acceptable salt
thereof or a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and a compound of the
disclosure or a pharmaceutically acceptable salt thereof.
[0131] Atopic dermatitis and other inflammatory skin diseases have
been associated with elevation of proinflammatory cytokines that
rely on the JAK-STAT pathway. Therefore, the compounds of the
disclosure, or a pharmaceutically acceptable salt thereof, may be
beneficial in a number of dermal inflammatory or pruritic
conditions that include, but are not limited to atopic dermatitis,
alopecia areata, vitiligo, psoriasis, dermatomyositis, cutaneous T
cell lymphoma (Netchiporouk et al., Cell Cycle. 2014; 13,
3331-3335) and subtypes (Sezary syndrome, mycosis fungoides,
pagetoid reticulosis, granulomatous slack skin, lymphomatoid
papulosis, pityriasis lichenoides chronica, pityriasis lichenoides
et varioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondary
cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30-
cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma,
Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric
lymphoma, blastic NK-cell lymphoma), prurigo nodularis, lichen
planus, primary localized cutaneous amyloidosis, bullous
pemphigoid, skin manifestations of graft versus host disease,
pemphigoid, discoid lupus, granuloma annulare, lichen simplex
chronicus, vulvar/scrotal/perianal pruritus, lichen sclerosus, post
herpetic neuralgia itch, lichen planopilaris, and foliculitis
decalvans. In particular, atopic dermatitis (Bao et al., JAK-STAT,
2013, 2, e24137), alopecia areata (Xing et al., Nat Med. 2014, 20,
1043-1049), vitiligo (Craiglow et al, JAMA Dermatol. 2015, 151,
1110-1112), prurigo nodularis (Sonkoly et al., J Allergy Clin
Immunol. 2006, 117, 411-417), lichen planus (Welz-Kubiak et al., J
Immunol Res. 2015, ID:854747), primary localized cutaneous
amyloidosis (Tanaka et al., Br J Dermatol. 2009, 161, 1217-1224),
bullous pemphigoid (Feliciani et al., Int J Immunopathol Pharmacol.
1999, 12, 55-61), and dermal manifestations of graft versus host
disease (Okiyama et al., J Invest Dermatol. 2014, 134, 992-1000)
are characterized by elevation of certain cytokines that signal via
JAK activation. Accordingly, compounds of the disclosure, or a
pharmaceutically acceptable salt thereof, may be able to alleviate
associated dermal inflammation or pruritus driven by these
cytokines. In particular, compounds of the disclosure, or a
pharmaceutically acceptable salt thereof, may be expected to be
useful for the treatment of atopic dermatitis and other
inflammatory skin diseases. In one aspect, therefore, the invention
provides a method of treating an inflammatory skin disease in a
mammal (e.g., a human), the method comprising applying a
pharmaceutical composition comprising a compound of the disclosure,
or a pharmaceutically acceptable salt thereof and a pharmaceutical
carrier to the skin of the mammal. In one aspect, the inflammatory
skin disease is atopic dermatitis.
[0132] Many ocular diseases have been shown to be associated with
elevations of proinflammatory cytokines that rely on the JAK-STAT
pathway. The compounds of the disclosure, or a pharmaceutically
acceptable salt thereof, therefore, may be useful for the treatment
of a number of ocular diseases that include, but are not limited
to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye
disease, age-related macular degeneration, and atopic
keratoconjunctivitis. In particular, uveitis (Horai and Caspi, J
Interferon Cytokine Res, 2011, 31, 733-744), diabetic retinopathy
(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12), diabetic
macular edema (Sohn et al., American Journal of Opthamology, 2011,
152, 686-694), dry eye disease (Stevenson et al, Arch Ophthalmol,
2012, 130, 90-100), and age-related macular degeneration
(Knickelbein et al, Int Ophthalmol Clin, 2015, 55(3), 63-78) are
characterized by elevation of certain pro-inflammatory cytokines
that signal via the JAK-STAT pathway. Accordingly, compounds of the
disclosure, or a pharmaceutically acceptable salt thereof, may be
able to alleviate the associated ocular inflammation and reverse
disease progression or provide symptom relief. In one aspect,
therefore, the invention provides a method of treating an ocular
disease in a mammal, the method comprising administering a
pharmaceutical composition comprising a compound of the disclosure
or a pharmaceutically-acceptable salt thereof and a pharmaceutical
carrier to the eye of the mammal. In one aspect, the ocular disease
is uveitis, diabetic retinopathy, diabetic macular edema, dry eye
disease, age-related macular degeneration, or atopic
keratoconjunctivitis. In one aspect, the method comprises
administering the compound of the disclosure, or a pharmaceutically
acceptable salt thereof by intravitreal injection. Compounds of the
disclosure, or a pharmaceutically acceptable salt thereof, may also
be used in combination with one or more compound useful to ocular
diseases.
[0133] The compounds of the disclosure, or a pharmaceutically
acceptable salt thereof, may also be useful to treat other diseases
such as other inflammatory diseases, autoimmune diseases or
cancers. The compounds of the disclosure, or a pharmaceutically
acceptable salt thereof, may be useful to treat one or more of
arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,
transplant rejection, xerophthalmia, psoriatic arthritis, diabetes,
insulin dependent diabetes, motor neurone disease, myelodysplastic
syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus
erythematosus, leukemia, chronic lymphocytic leukemia, chronic
myelocytic leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-cell
lymphoma, hepatocellular carcinoma, Hodgkins disease, breast
cancer, Multiple myeloma, melanoma, non-Hodgkin lymphoma,
non-small-cell lung cancer, ovarian clear cell carcinoma, ovary
tumor, pancreas tumor, polycythemia vera, Sjoegrens syndrome, soft
tissue sarcoma, sarcoma, splenomegaly, T-cell lymphoma, and
thalassemia major.
[0134] Combination Therapy
[0135] Compounds of the disclosure or a pharmaceutically acceptable
salt thereof may be used in combination with one or more agents
which act by the same mechanism or by different mechanisms to treat
a disease. The different agents may be administered sequentially or
simultaneously, in separate compositions or in the same
composition. Useful classes of agents for combination therapy
include, but are not limited to, a beta 2 adrenoceptor agonist, a
muscarinic receptor antagonist, a glucocorticoid agonist, a
G-protein coupled receptor-44 antagonist, a leukotriene D4
antagonist, a muscarinic M3 receptor antagonist, a histamine H1
receptor antagonist, an immunoglobulin E antagonist, a PDE 4
inhibitor, an IL-4 antagonist, a muscarinic M1 receptor antagonist,
a histamine receptor antagonist, an IL-13 antagonist, an IL-5
antagonist, a 5-Lipoxygenase inhibitor, a beta adrenoceptor
agonist, a CCR3 chemokine antagonist, a CFTR stimulator, an
immunoglobulin modulator, an interleukin 33 ligand inhibitor, a PDE
3 inhibitor, a phosphoinositide-3 kinase delta inhibitor, a
thromboxane A2 antagonist, an elastase inhibitor, a Kit tyrosine
kinase inhibitor, a leukotriene E4 antagonist, a leukotriene
antagonist, a PGD2 antagonist, a TNF alpha ligand inhibitor, a TNF
binding agent, a complement cascade inhibitor, an eotaxin ligand
inhibitor, a glutathione reductase inhibitor, an histamine H4
receptor antagonist, an IL-6 antagonist, an IL2 gene stimulator, an
immunoglobulin gamma Fc receptor IIB modulator, an interferon gamma
ligand, an interleukin 13 ligand inhibitor, an interleukin 17
ligand inhibitor, a L-Selectin antagonist, a leukocyte elastase
inhibitor, a leukotriene C4 antagonist, a Leukotriene C4 synthase
inhibitor, a membrane copper amine oxidase inhibitor, a
metalloprotease-12 inhibitor, a metalloprotease-9 inhibitor, a mite
allergen modulator, a muscarinic receptor modulator, a nicotinic
acetylcholine receptor agonist, a nuclear factor kappa B inhibitor,
a p-Selectin antagonist, a PDE 5 inhibitor, a PDGF receptor
antagonist, a phosphoinositide-3 kinase gamma inhibitor, a TLR-7
agonist, a TNF antagonist, an Abl tyrosine kinase inhibitor, an
acetylcholine receptor antagonist, an acidic mammalian chitinase
inhibitor, an ACTH receptor agonist, an actin polymerization
modulator, an adenosine A1 receptor antagonist, an adenylate
cyclase stimulator, an adrenoceptor antagonist, an
adrenocorticotrophic hormone ligand, an alcohol dehydrogenase 5
inhibitor, an alpha 1 antitrypsin stimulator, an alpha 1 proteinase
inhibitor, an androgen receptor modulator, an angiotensin
converting enzyme 2 stimulator, an ANP agonist, a Bcr protein
inhibitor, a beta 1 adrenoceptor antagonist, a beta 2 adrenoceptor
antagonist, a beta 2 adrenoceptor modulator, a beta amyloid
modulator, a BMP10 gene inhibitor, a BMP15 gene inhibitor, a
calcium channel inhibitor, a cathepsin G inhibitor, a CCL26 gene
inhibitor, a CCR3 chemokine modulator, a CCR4 chemokine antagonist,
a cell adhesion molecule inhibitor, a chaperonin stimulator, a
chitinase inhibitor, a collagen I antagonist, a complement C3
inhibitor, a CSF-1 antagonist, a CXCR2 chemokine antagonist, a
cytokine receptor common beta chain modulator, a cytotoxic
T-lymphocyte protein-4 stimulator, a deoxyribonuclease I
stimulator, a deoxyribonuclease stimulator, a dipeptidyl peptidase
I inhibitor, a DNA gyrase inhibitor, a DP prostanoid receptor
modulator, an E-Selectin antagonist, an EGFR family tyrosine kinase
receptor inhibitor, an elastin modulator, an Endothelin ET-A
antagonist, an Endothelin ET-B antagonist, an epoxide hydrolase
inhibitor, a FGF3 receptor antagonist, a Fyn tyrosine kinase
inhibitor, a GATA 3 transcription factor inhibitor, a
Glucosylceramidase modulator, a Glutamate receptor modulator, a
GM-CSF ligand inhibitor, a Guanylate cyclase stimulator, a H+ K+
ATPase inhibitor, an hemoglobin modulator, an Heparin agonist, an
Histone deacetylase inhibitor, an Histone deacetylase-2 stimulator,
an HMG CoA reductase inhibitor, an I-kappa B kinase beta inhibitor,
an ICAM1 gene inhibitor, an IL-17 antagonist, an IL-17 receptor
modulator, an IL-23 antagonist, an IL-4 receptor modulator, an
Immunoglobulin G modulator, an Immunoglobulin G1 agonist, an
Immunoglobulin G1 modulator, an Immunoglobulin epsilon Fc receptor
IA antagonist, an Immunoglobulin gamma Fc receptor IIB antagonist,
an Immunoglobulin kappa modulator, an Insulin sensitizer, an
Interferon beta ligand, an Interleukin 1 like receptor antagonist,
an Interleukin 18 ligand inhibitor, an Interleukin receptor 17A
antagonist, an Interleukin-1 beta ligand inhibitor, an
Interleukin-5 ligand inhibitor, an Interleukin-6 ligand inhibitor,
a KCNA voltage-gated potassium channel-3 inhibitor, a Kit ligand
inhibitor, a Laminin-5 agonist, a Leukotriene CysLT1 receptor
antagonist, a Leukotriene CysLT2 receptor antagonist, a LOXL2 gene
inhibitor, a Lyn tyrosine kinase inhibitor, a MARCKS protein
inhibitor, a MDR associated protein 4 inhibitor, a
Metalloprotease-2 modulator, a Metalloprotease-9 modulator, a
Mineralocorticoid receptor antagonist, a Muscarinic M2 receptor
antagonist, a Muscarinic M4 receptor antagonist, a Muscarinic M5
receptor antagonist, a Natriuretic peptide receptor A agonist, a
Natural killer cell receptor modulator, a Nicotinic ACh receptor
alpha 7 subunit stimulator, a NK cell receptor modulator, a Nuclear
factor kappa B modulator, an opioid growth factor receptor agonist,
a P-Glycoprotein inhibitor, a P2X3 purinoceptor antagonist, a p38
MAP kinase inhibitor, a Peptidase 1 modulator, a phospholipase A2
inhibitor, a phospholipase C inhibitor, a plasminogen activator
inhibitor 1 inhibitor, a platelet activating factor receptor
antagonist, a PPAR gamma agonist, a prostacyclin agonist, a protein
tyrosine kinase inhibitor, a SH2 domain inositol phosphatase 1
stimulator, a signal transduction inhibitor, a sodium channel
inhibitor, a STAT-3 modulator, a Stem cell antigen-1 inhibitor, a
superoxide dismutase modulator, a T cell surface glycoprotein CD28
inhibitor, a T-cell surface glycoprotein CD8 inhibitor, a TGF beta
agonist, a TGF beta antagonist, a thromboxane synthetase inhibitor,
a thymic stromal lymphoprotein ligand inhibitor, a thymosin
agonist, a thymosin beta 4 ligand, a TLR-8 agonist, a TLR-9
agonist, a TLR9 gene stimulator, a Topoisomerase IV inhibitor, a
Troponin I fast skeletal muscle stimulator, a Troponin T fast
skeletal muscle stimulator, a Type I IL-1 receptor antagonist, a
Type II TNF receptor modulator, an ion channel modulator, a
uteroglobin stimulator, and a VIP agonist.
[0136] Specific agents that may be used in combination with the
present JAK inhibitor compounds include, but are not limited to
rosiptor acetate, umeclidinium bromide, secukinumab, metenkefalin
acetate, tridecactide acetate, fluticasone propionate,
alpha-cyclodextrin-stabilized sulforaphane, tezepelumab, mometasone
furoate, BI-1467335, dupilumab, aclidinium, formoterol, AZD-1419,
HI-1640V, rivipansel, CMP-001, mannitol, ANB-020, omalizumab,
tregalizumab, Mitizax, benralizumab, golimumab, roflumilast,
imatinib, REGN-3500, masitinib, apremilast, RPL-554, Actimmune,
adalimumab, rupatadine, parogrelil, MK-1029, beclometasone
dipropionate, formoterol fumarate, mogamulizumab, seratrodast,
UCB-4144, nemiralisib, CK-2127107, fevipiprant, danirixin,
bosentan, abatacept, EC-18, duvelisib, dociparstat, ciprofloxacin,
salbutamol HFA, erdosteine, PrEP-001, nedocromil, CDX-0158,
salbutamol, enobosarm, R-TPR-022, lenzilumab, fluticasone furoate,
vilanterol trifenatate, fluticasone propionate, salmeterol, PT-007,
PRS-060, remestemcel-L, citrulline, RPC-4046, nitric oxide, DS-102,
gerilimzumab, Actair, fluticasone furoate, umeclidinium,
vilanterol, AG-NPP709, Gamunex, infliximab, Ampion, acumapimod,
canakinumab, INS-1007, CYP-001, sirukumab, fluticasone propionate,
mepolizumab, pitavastatin, solithromycin, etanercept, ivacaftor,
anakinra, MPC-300-IV, glycopyrronium bromide, aclidinium bromide,
FP-025, risankizumab, glycopyrronium, formoterol fumarate,
Adipocell, YPL-001, tiotropium bromide, glycopyrronium bromide,
indacaterol maleate, andecaliximab, olodaterol, esomeprazole, dust
mite vaccine, mugwort pollen allergen vaccine, vamorolone,
gefapixant, revefenacin, gefitinib, Rejoin, tipelukast,
bedoradrine, SCM-CGH, SHP-652, RNS-60, brodalumab, BIO-11006,
umeclidinium bromide, vilanterol trifenatate, ipratropium bromide,
tralokinumab, PUR-1800, VX-561, VX-371, olopatadine, tulobuterol,
formoterol fumarate, triamcinolone acetonide, reslizumab,
salmeterol xinafoate, fluticasone propionate, beclometasone
dipropionate, formoterol fumarate, tiotropium bromide, ligelizumab,
RUTI, bertilimumab, omalizumab, glycopyrronium bromide, SENS-111,
beclomethasone dipropionate, CHF-5992, LT-4001, indacaterol,
glycopyrronium bromide, mometasone furoate, fexofenadine,
glycopyrronium bromide, azithromycin, AZD-7594, formoterol,
CHF-6001, batefenterol, OATD-01, olodaterol, CJM-112,
rosiglitazone, salmeterol, setipiprant, inhaled interferon beta,
AZD-8871, plecanatide, fluticasone, salmeterol, eicosapentaenoic
acid monoglycerides, lebrikizumab, RG-6149, QBKPN, Mometasone,
indacaterol, AZD-9898, sodium pyruvate, zileuton, CG-201,
imidafenacin, CNTO-6785, CLBS-03, mometasone, RGN-137, procaterol,
formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone,
MV-130, GC-1112, Allergovac depot , MEDI-3506, QBW-251, ZPL-389,
udenafil, GSK-3772847, levocetirizine, AXP-1275, ADC-3680,
timapiprant, abediterol, AZD-7594, ipratropium bromide, salbutamol
sulfate, tadekinig alfa, ACT-774312, dornase alfa, iloprost,
batefenterol, fluticasone furoate, alicaforsen, ciclesonide,
emeramide, arformoterol, SB-010, Ozagrel, BTT-1023, Dectrekumab,
levalbuterol, pranlukast, hyaluronic acid, GSK-2292767, Formoterol,
NOV-14, Lucinactant, salbutamol, prednisolone, ebastine,
dexamethasone cipecilate, GSK-2586881, BI-443651, GSK-2256294,
VR-179, VR-096, hdm-ASIT+, budesonide, GSK-2245035, VTX-1463,
Emedastine, dexpramipexole, levalbuterol, N-6022, dexamethasone
sodium phosphate, PIN-201104, OPK-0018, TEV-48107, suplatast,
BI-1060469, Gemilukast, interferon gamma, dalazatide, bilastine,
fluticasone propionate, salmeterol xinafoate, RP-3128,
bencycloquidium bromide, reslizumab, PBF-680, CRTH2 antagonist,
Pranlukast, salmeterol xinafoate, fluticasone propionate,
tiotropium bromide monohydrate, masilukast, RG-7990, Doxofylline,
abediterol, glycopyrronium bromide, TEV-46017, ASM-024, fluticasone
propionate, glycopyrronium bromide, salmeterol xinafoate,
salbutamol, TA-270, Flunisolide, sodium chromoglycate, Epsi-gam,
ZPL-521, salbutamol, aviptadil, TRN-157, Zafirlukast, Stempeucel,
pemirolast sodium, nadolol, fluticasone propionate+salmeterol
xinafoate, RV-1729, salbutamol sulfate, carbon
dioxide+perfluorooctyl bromide, APL-1, dectrekumab+VAK-694, lysine
acetylsalicylate, zileuton, TR-4, human allogenic adipose-derived
mesenchymal progenitor cell therapy, MEDI-9314, PL-3994, HMP-301,
TD-5471, NKTT-120, pemirolast, beclomethasone dipropionate,
trantinterol, monosodium alpha luminol, IMD-1041, AM-211, TBS-5,
ARRY-502, seratrodast, recombinant midismase, ASM-8, deflazacort,
bambuterol, RBx-10017609, ipratropium+fenoterol,
fluticasone+formoterol, epinastine, WIN-901X,
VALERGEN-DS,OligoG-COPD-5/20, tulobuterol, oxis Turbuhaler,
DSP-3025, ASM-024, mizolastine, budesonide +salmeterol, LH-011,
AXP-E, histamine human immunoglobulin, YHD-001, theophylline,
ambroxol+erdosteine, ramatroban, montelukast, pranlukast,
AG-1321001, tulobuterol, ipratropium+salbutamol, tranilast,
methylprednisolone suleptanate, colforsin daropate, repirinast, and
doxofylline.
[0137] Also provided, herein, is a pharmaceutical composition
comprising a compound of the disclosure or a pharmaceutically
acceptable salt thereof and one or more other therapeutic agents.
The therapeutic agent may be selected from the class of agents
specified above and from the list of specific agent described
above. In some embodiments, the pharmaceutical composition is
suitable for delivery to the lungs. In some embodiments, the
pharmaceutical composition is suitable for inhaled or nebulized
administration. In some embodiments, the pharmaceutical composition
is a dry powder or a liquid composition.
[0138] Further, in a method aspect, the invention provides a method
of treating a disease or disorder in a mammal comprising
administering to the mammal a compound of the disclosure or a
pharmaceutically acceptable salt thereof and one or more other
therapeutic agents.
[0139] When used in combination therapy, the agents may be
formulated in a single pharmaceutical composition, or the agents
may be provided in separate compositions that are administered
simultaneously or at separate times, by the same or by different
routes of administration. Such compositions can be packaged
separately or may be packaged together as a kit. The two or more
therapeutic agents in the kit may be administered by the same route
of administration or by different routes of administration.
[0140] Compounds of the invention have been demonstrated to be
potent inhibitors of the JAK1, JAK2, JAK3, and TYK2 enzymes in
enzyme binding assays, to have potent functional activity without
cytotoxicity in cellular assays, and to exert the pharmacodynamic
effects of JAK inhibition in preclinical models, as described in
the following examples.
EXAMPLES
[0141] The following synthetic and biological examples are offered
to illustrate the invention, and are not to be construed in any way
as limiting the scope of the invention. In the examples below, the
following abbreviations have the following meanings unless
otherwise indicated. Abbreviations not defined below have their
generally accepted meanings.
[0142] ACN=acetonitrile
[0143] DCM=dichloromethane
[0144] DIPEA=N,N-diisopropylethylamine
[0145] DMF=N,N-dimethylformamide
[0146] EtOAc=ethyl acetate
[0147] h=hour(s)
[0148] HATU=N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate
[0149] IPA=isopropyl alcohol
[0150] IPAc=isopropylacetate
[0151] MeOH=methanol
[0152] min=minute(s)
[0153]
Pd(PPh.sub.3).sub.4=tetrakis(triphenylphosphine)palladium(0)
[0154] RT=room temperature
[0155] TFA=trifluoroacetic acid
[0156] THF=tetrahydrofuran
[0157]
bis(pinacolato)diboron=4,4,5,5,4',4',5',5'-octamethyl-[2,2']bi[[1,3-
,2]dioxaborolanyl]Reagents and solvents were purchased from
commercial suppliers (Aldrich, Fluka, Sigma, etc.), and used
without further purification. Progress of reaction mixtures was
monitored by thin layer chromatography (TLC), analytical high
performance liquid chromatography (anal. HPLC), and mass
spectrometry. Reaction mixtures were worked up as described
specifically in each reaction; commonly they were purified by
extraction and other purification methods such as temperature-, and
solvent-dependent crystallization, and precipitation. In addition,
reaction mixtures were routinely purified by column chromatography
or by preparative HPLC, typically using C18 or BDS column packings
and conventional eluents. Typical preparative HPLC conditions are
described below.
[0158] Characterization of reaction products was routinely carried
out by mass and .sup.1H-NMR spectrometry. For NMR analysis, samples
were dissolved in deuterated solvent (such as CD.sub.3OD,
CDCl.sub.3, or d.sub.6-DMSO), and .sup.1H-NMR spectra were acquired
with a Varian Gemini 2000 instrument (400 MHz) under standard
observation conditions. Mass spectrometric identification of
compounds was performed by an electrospray ionization method (ESMS)
with an Applied Biosystems (Foster City, Calif.) model API 150 EX
instrument or a Waters (Milford, Mass.) 3100 instrument, coupled to
autopurification systems.
Preparative HPLC Conditions
[0159] Column: C18, 5 .mu.m. 21.2.times.150 mm or C18, 5 .mu.m
21.times.250 or C14, 5 .mu.m 21.times.150 mm
[0160] Column temperature: Room Temperature
[0161] Flow rate: 20.0 mL/min
[0162] Mobile Phases: A=Water+0.05% TFA
[0163] B=ACN+0.05% TFA,
[0164] Injection volume: (100-1500 .mu.L)
[0165] Detector wavelength: 214 nm
[0166] Crude compounds were dissolved in 1:1 water:acetic acid at
about 50 mg/mL . A 4 minute analytical scale test run was carried
out using a 2.1.times.50 mm C18 column followed by a 15 or 20
minute preparative scale run using 100 .mu.L injection with the
gradient based on the % B retention of the analytical scale test
run. Exact gradients were sample dependent. Samples with close
running impurities were checked with a 21.times.250 mm C18 column
and/or a 21.times.150 mm C14 column for best separation. Fractions
containing desired product were identified by mass spectrometric
analysis.
[0167] Preparation 1:
2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxab-
orolane (9)
##STR00017##
[0168] (a) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (21)
[0169] To a solution of 4-bromo-5-ethyl-2-fluorophenol (20) (20 g,
910.32 mmol) in ACN (250 mL) was added K.sub.2CO.sub.3 (31.55 g,
228.3 mmol) followed by benzyl bromide (13.10 mL, 109.58 mmol) drop
wise. The resulting reaction mixture was stirred at 80.degree. C.
for 2 h. The aqueous layer was extracted with EtOAc (three times),
combined and washed with brine. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated under reduced pressure to afford
the title intermediate as a pale yellow oily liquid (25 g, 89%
yield). .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.48-7.30 (m,
5H), 7.27 (d, J=10.5 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 5.12 (s, 2H),
2.66 (q, J=7.5 Hz, 2H), 1.16 (t, J=7.5 Hz, 3H).
[0170] (b)
2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,-
3,2-dioxaborolane (9)
[0171] To a solution of the product of the previous step (21) (12.5
g, 40.45 mmol) in dioxane (100 mL) was added bis(pinacolato)diboron
(15.40 g, 60.67 mmol) and KOAc (11.9 g, 121.35 mmol). The reaction
mixture was purged with nitrogen for 15 min followed by addition of
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1.65 g, 2.023 mmol). The resulting
reaction mixture was stirred and heated at 110.degree. C. for 3 h,
filtered through Celite and the residue washed with EtOAc. The
filtrate was diluted with excess EtOAc (200 mL) and washed with
water (100 mL) followed by brine (100 mL), dried over sodium
sulfate and concentrated in vacuo to get crude product which was
purified by column chromatography over (100-200) silica gel, eluted
with 3-5% EtOAc: hexane to afford the desired product as an
off-white solid (9.50 g, 66% yield). .sup.1H NMR (400 MHz,
chloroform-d) .delta. 7.54-7.27 (m, 6H), 6.81 (d, J=7.9 Hz, 1H),
5.16 (s, 2H), 2.84 (q, J=7.5 Hz, 2H), 1.32 (s, 12H), 1.14 (t, J=7.5
Hz, 3H).
[0172] Preparation 2:
6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(-
trimethylstannyl)-1H-indazole (3')
##STR00018## ##STR00019##
[0173] (a)
6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-
-2-yl)-1H-indazole (22)
[0174] To a solution of
6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (10) (50 g, 178.57
mmol) and
2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxab-
orolane (9) (76.3 g, 214.29 mmol) in DMF:H.sub.2O (480:120 mL) was
added K.sub.3PO.sub.4 (94.64 g, 446.86 mmol). The reaction mixture
was degassed with nitrogen for 15 min, then
Pd(PPh.sub.3).sub.2Cl.sub.2 catalyst (6.26 g, 8.93 mmol) was added
and the mixture was again degassed with nitrogen for 5 min stirred,
and heated at 100-110.degree. C. for 5 h. The reaction mixture was
filtered through Celite and the residue was washed with EtOAc. The
filtrate was diluted with EtOAc, washed with cold water and brine,
dried over sodium sulfate and concentrated in vacuo to provide
crude product which was purified by flash column chromatography to
afford the title intermediate as a white solid (65 g, 86% yield).
(m/z): [M+H].sup.+ calcd for C.sub.27H.sub.27FN.sub.2O.sub.2 431.21
found 431.46. .sup.1H NMR (400 MHz, chloroform-d) .delta. 8.06-7.98
(m, 2H), 7.70 (d, J=8.2 Hz, 1H), 7.51-7.32 (m, 5H), 7.08 (dd,
J=809.6, 8.3 Hz, 1H), 7.03 (d, J=11.9 Hz, 1H), 6.95 (d, J=8.5 Hz,
1H), 5.76-5.64 (m, 1H), 5.20 (s, 2H), 4.04 (d, J=10.1 Hz, 1H), 3.72
(t, J=9.7 Hz, 1H), 2.52 (q, J=7.5 Hz, 2H), 2.22-2.02 (m, 3H),
1.80-1.71 (m, 3H), 1.06 (t, J=7.5 Hz, 3H).
[0175] (b) 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazole
(23)
[0176] To a solution of the product of the previous step (22) (65
g, 151.16 mmol) in methanol (700 mL) was added conc. HCl (120 mL)
and the resulting solution was heated at 60-65.degree. C. for 3 h,
cooled to RT, and concentrated in vacuo. The residue was dissolved
in EtOAc and washed with saturated NaHCO.sub.3 aqueous solution and
water. The organic layer was dried over anhydrous Na.sub.2SO.sub.4
and concentrated in vacuo to afford the title intermediate as a
white solid (52 g, 99% (crude)). .sup.1H NMR (400 MHz,
chloroform-d) .delta. 8.13 (s, 1H), 7.77 (d, J=8.3 Hz, 1H),
7.59-7.30 (m, 6H), 7.10 (d, J=8.3 Hz, 1H), 7.01 (d, J=11.8 Hz, 1H),
6.96 (d, J=8.4 Hz, 1H), 5.21 (s, 2H), 2.53 (q, J=7.5 Hz, 2H), 1.05
(t, J=7.5 Hz, 3H).
[0177] (c)
6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1H-indazole
(24)
[0178] To a solution of
6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazole (23) (56 g,
161.18 mmol) in DMF (400 mL) was added KOH (36.2 g, 647.39 mmol)
and the mixture was stirred for 5 min. A solution of iodine (82.2
g, 323.69 mmol) in DMF (100 mL) was added slowly at 0.degree. C.
and stirred at RT for 30 min, diluted with water (3.times.150 mL)
and extracted with EtOAc (3.times.200 mL). The organic layer was
washed with saturated sodium metabisulfite aqueous solution
(3.times.200 mL) and water (400 mL), dried over anhydrous
Na.sub.2SO.sub.4 and concentrated under reduced pressure to get
crude product which was purified by flash column chromatography to
afford the title intermediate as a brownish semi-solid (64 g, 84%
yield). .sup.1H NMR (400 MHz, chloroform-d) .delta. 10.49 (s, 1H),
7.57-7.32 (m, 7H), 7.16 (d, J=8.3 Hz, 1H), 7.04-6.91 (m, 2H), 5.20
(s, 2H), 2.51 (q, J=7.4 Hz, 2H), 1.04 (t, J=7.5 Hz, 3H).
[0179] (d)
6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1-(tetrahydro-2-
H-pyran-2-yl)-.sup.1H-indazole (25)
[0180] To an ice-cold solution of the product of the previous step
(24) (60 g, 127.12 mmol) in DCM (700 mL) was addedp-toluensulfonic
acid (4.84 g, 25.423 mmol) followed by 3,4-dihydro-2H-pyran (17.43
mL, 190.68 mmol) drop wise. The reaction mixture was stirred at RT
overnight, diluted with DCM and washed with saturated NaHCO.sub.3
aqueous solution and brine. The organic layer was dried over
anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure
to provide crude product which was purified by flash chromatography
(silica gel) to afford the title intermediate as an off white solid
(64 g, 91% yield). (m/z): [M+H].sup.+ calcd for
C.sub.27H.sub.26FIN.sub.2O.sub.2 557.10 found 557.30. .sup.1H NMR
(400 MHz, chloroform-d) .delta. 7.56-7.31 (m, 7H), 7.14 (d, J=8.3
Hz, 1H), 7.01 (d, J =11.8 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H), 5.68 (d,
J=9.3 Hz, 1H), 5.20 (s, 2H), 4.08-3.99 (m, 1H), 3.77-3.64 (m, 1H),
2.50 (q, J=7.2 Hz, 2H), 2.23-1.97 (m, 3H), 1.81-1.68 (m, 3H), 1.06
(t, J=7.4 Hz, 3H).
[0181] (e)
6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-
-2-yl)-3-(trimethylstarmyl)-1H-indazole (3')
[0182] To a solution of
6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1-(tetrahydro-2H-pyran-2--
yl)-1H-indazole (25) (20 g, 35.97 mmol) in toluene (150 mL) was
added hexamethylditin (9.2 mL, 43.17 mmol). The reaction mixture
was degassed with nitrogen for 20 min followed by addition of
tetrakis (2.0 g, 1.80 mmol) and then stirred at 100.degree. C. for
2 h, cooled to RT, filtered through Celite and residue washed with
EtOAc The filtrate was concentrated and purified by column
chromatography (over neutral alumina), eluted with 2-5%.
EtOAc:hexane to afford the title compound (17.50 g, 82% yield).
(m/z): [M+H].sup.+ calcd for C.sub.27H.sub.26FIN.sub.2O.sub.2
557.10 found 557.30. (m/z): [M+H].sup.+ calcd for
C.sub.30H.sub.35FN.sub.2O.sub.2Sn 595.17, 593.17 found 595.49,
593.55. .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.68 (d, J=8.0
Hz, 1H), 7.57-7.29 (m, 6H), 7.13-7.00 (m, 2H), 6.96 (d, J=8.4 Hz,
1H), 5.81-5.68 (m, 1H), 5.21 (s, 2H), 4.13-4.00 (m, 1H), 3.81-3.66
(m, 1H), 2.54 (q, J=7.3 Hz, 2H), 2.23-2.00 (m, 2H), 1.87-1.59 (m,
4H), 1.08 (t, J=7.5 Hz, 3H), 0.47 (s, 9H).
[0183] Preparation 3: 5-(tert-butyl) 6-methyl
(S)-2-iodo-3-((2-trimethylsilyl)ethoxy)
methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(4')
##STR00020##
[0184] (a)
(S)-4,5,6,7-Tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid
(11)
[0185] To a stirred suspension of L-histidine (26) (50 g, 322.24
mmol) in water (420 mL) was added conc. HCl (29 mL) drop wise at
0.degree. C. followed by formaldehyde (55 mL, 676.72 mmol) in one
portion at 0.degree. C. The resulting reaction mixture was stirred
for 30 min and then heated at 75.degree. C. for 6 h and
concentrated. The resulting crude was stirred for 2 h with diethyl
ether, filtered and washed with IPA:THF (100:300 mL) to provide the
HCl salt of the title intermediate as an off white solid (75 g 99%
yield (crude)). (m/z): [M+H].sup.+ calcd for
C.sub.7H.sub.9N.sub.3O.sub.2 168.07 found 168.17.
[0186] (b) Methyl
(S)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylate
(27)
[0187] To a stirred solution of the product of the previous step
(11) (75.0 g, 312.5 mmol) in methanol (1500 mL) was added
SOCl.sub.2 (45.6 mL, 625 mmol) dropwise at 0.degree. C. and stirred
at RT for 16 h, then heated up to reflux (70.degree. C.) for 1 h.
The solvent was removed by distillation and the crude product was
triturated with methanol followed by diethyl ether to provide the
crude HCl salt of the title intermediate as an off white solid (80
g crude). .sup.1H NMR (400 MHz, DMSO-d6) .delta.9.05 (s, 1H), 4.71
(dd, J=9.4, 5.2 Hz, 1H), 4.36 (d, J=15.5 Hz, 1H), 4.30 (d, J=15.6
Hz, 1H), 3.82 (s, 3H), 3.44-3.21 (m, 2H).
[0188] (c) 5 -(tert-Butyl) 6-methyl
(S)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(28)
[0189] To a stirred solution of the product of the previous step
(27) (80.0 g, 314.96 mmol) in methanol (1000 mL) was added DIPEA
(282 mL, 1574 mmol) followed by di-tent-butyl dicarbonate (172 mL,
787.48 mmol) at 0.degree. C. The reaction mixture was stirred at RT
for 16 h and then liquid NH.sub.3 (150 mL, 25% in water) was added
and the reaction mixture was stirred again for 16 h at RT, methanol
was removed by distillation and the residue was extracted in DCM
(3.times.200 mL). Combined organic extracts were dried over
anhydrous Na.sub.2SO.sub.4, concentrated and purified by flash
chromatography (100-200 mesh silica gel), eluted with 5% MeOH:DCM
to afford the title intermediate (41 g, 46%. yield). (m/z):
[M+H].sup.+ calcd for C.sub.13H.sub.19N.sub.3O.sub.4 282.14 found
282.21. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.85 (s, 1H), 7.50
(s, 1H), 5.18 (dd, J=49.3, 5.1 Hz, 1H), 4.51 (t, J=14.2 Hz, 1H),
4.09 (dd, J=43.9, 16.1 Hz, 1H), 3.59 (s, 3H), 3.08 (d, J=15.5 Hz,
1H), 2.94 (d, J=15.1 Hz, 1H), 1.45 (s, 9H).
[0190] (d) 5-(tent-Butyl) 6-methyl
(S)-2-iodo-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(29)
[0191] To a solution of the product of the previous step (29) (41.0
g, 145.9 mmol) in THF (500 mL) was added N-iodosuccinimide (66.0 g,
291.8 mmol) at 0.degree. C. and the resulting solution was stirred
at RT for 4 h, diluted with water and extracted with ethyl acetate.
The organic portion was washed with 10% sodium thiosulphate
solution (3.times.200 mL). The combined organic layer was dried
over anhydrous sodium sulfate, and concentrated to provide the
title compound 60 g (crude),which was used in the next step without
further purification. (m/z): [M+H].sup.+ calcd for
C.sub.13H.sub.18IN.sub.3O.sub.4 408.03 found 408.31. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 12.48 (s, 1H), 5.34-4.97 (m, 1H),
4.67-4.35 (m, 1H), 4.12-3.95 (m, 1H), 3.60 (s, 3H), 3.14-2.82 (m,
2H), 1.44 (s, 9H).
[0192] (e) 5-(tert-Butyl) 6-methyl
(S)-2-iodo-3-((2-trimethylsilyl)ethoxy)
methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(4')
[0193] To a stirred solution of 5-(tent-butyl) 6-methyl
(S)-2-iodo-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(29) (40 g, 0.098 mol) in DMF (150 mL) was added DIPEA (35.1 mL,
0.19 mol) at 0.degree. C. The reaction mixture was stirred for 10
min then 2-(trimethylsilyl)-ethoxymethyl chloride (19.1 mL, 0.10
mol) was added drop wise at 0.degree. C. The resulting reaction
mixture was stirred for 3 h at RT. After 4 h chilled water was
added and the reaction mixture was extracted with EtOAc
(2.times.200 mL). The organic layer was dried over anhydrous sodium
sulphate, concentrated, and purified by flash column
chromatography, eluted with 20-35% EtOAc:hexane, to afford the
title product as a pale yellow viscous liquid (27 g). (m/z):
[M+H].sup.+ calcd for C.sub.19H.sub.32IN.sub.3O.sub.5Si 538.12
found 538.42. .sup.1H NMR (400 MHz, DMSO-d6) .delta.5.33-5.04 (m,
3H), 4.79-4.56 (m, 1H), 4.54-4.14 (m, 1H), 3.60 (s, 3H), 3.47 (t,
J=7.8 Hz, 2H), 3.31-3.16 (m, 1H), 2.97 (t, J=18.9 Hz, 1H), 1.44 (s,
9H), 0.92-0.74 (m, 2H), -0.03 (s, 9H).
[0194] Preparation 4:
(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1-(t-
etrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-
(trimethylsilypethoxy)methyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-
e-6-carboxylic acid (7')
##STR00021##
[0195] (a) 5-(tent-Butyl) 6-methyl
(6S)-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-
-yl)-1H-indazol-3-yl)-3-((2- (trimethylsilyl) ethoxy)
methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(5')
[0196] To a stirred solution of 5-(tent-butyl) 6-methyl
(S)-2-iodo-3-((2-trimethylsilyl)ethoxy)
methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate
(4') (17.0 g, 31.65 mmol) in toluene (500 mL) was added
6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(-
trimethylstarmyl)-1H-indazole (3') (20 g, 34.82 mmol). The reaction
mixture was purged with argon for 15 min, Pd(PPh3)4 (3.6 g, 3.16
mmol) and copper iodide (1.20 g, 6.33 mmol) were added and the
reaction mixture was stirred at 120.degree. C. for 16 h. The
reaction mixture was filtered through
[0197] Celite, the filtrate was concentrated under reduced pressure
and purified by silica gel column chromatography (Redisep 80 g
column, eluted with DCM for 10 min and then 15-20% EtOAc in Hexane
to afford the title intermediate as a yellow solid (15.10 g, 58%
yield). (m/z): [M+H].sup.+ calcd for
C.sub.46H.sub.58FN.sub.5O.sub.7Si 840.41 found 840.54. .sup.1H NMR
(400 MHz, Chloroform-d) .delta. 8.43 (s, 1H), 7.54-7.33 (m, 6H),
7.20 (s, 1H), 7.05 (d, J=11.4 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H),
6.09-5.69 (m, 3H), 5.59-5.36 (m, 1H), 5.20 (s, 2H), 4.97-4.80 (m,
1H), 4.12-3.90 (m, 1H), 3.68 (s, 3H), 3.57-3.47 (m, 2H), 3.40 (d,
1H), 3.21-3.05 (m, 1H), 2.74-2.34 (m, 4H), 2.25-2.07 (m, 2H),
1.94-1.65 (m, 4H), 1.54 (s, 9H), 1.12-0.99 (m, 3H), 0.91-0.75 (m,
2H), -0.12 (s, 9H).
[0198] (b) 6-Benzyl 5-(tent-butyl)
(6S)-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-
-yl)-1H-indazol-3-yl)-3-((2-
(trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridi-
ne-5,6-dicarboxylate (6')
[0199] To a round bottom flask was added the product of the
previous step (5') (15.0 g, 17.85 mmol) in toluene (400 mL), benzyl
alcohol (46.3 mL) and Ti(OEt)4 (7.15 mL, 35.70 mmol) and the
reaction mixture was refluxed vigorously (140.degree. C.) for 48 h,
diluted with water and extracted with DCM. The suspension was
filtered, filtrate was dried over Na.sub.2SO.sub.4, concentrated
under reduced pressure and purified by silica gel column
chromatography (Redisep 80 g column, 0-5% EtOAc in hexanes) for 20
min to remove excess benzyl alcohol, then eluted with 10-15% EtOAc
in Hexane) to provide the title intermediate. .sup.1H NMR
consistent with structure. (m/z): [M+H].sup.+ calcd for
C.sub.52H.sub.62FN.sub.5O.sub.7Si 916.44 found 916.86.
[0200] (c)
(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyph-
enyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-
(trimethylsilypethoxy)methyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-
e-6-carboxylic acid (7')
[0201] To a stirred solution of the product of the previous step
(6') (21.0 g, 22.92 mmol) in 1:1 IPA:THF (400 mL)) was added
Pd(OH).sub.2 (5.0 g). The reaction mixture was stirred at RT for 16
h under a hydrogen balloon, filtered through Celite, concentrated
under reduced pressure, and purified by silica gel column
chromatography (Redisep 80 g column, eluted with 25-40% EtOAc in
Hexane) to provide the title compound (6.1 g, 8.29 mmol) as an
off-white solid). (m/z): [M+H].sup.+ calcd for
C38H.sub.50FN.sub.5O.sub.7Si 736.35 found 736.5. .sup.1H NMR
consistent with structure. (m/z): [M+H].sup.+ calcd for
C.sub.38H.sub.50FN.sub.5O.sub.7Si 736.35 found 736.5. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 12.94 (s, 1H), 9.86 (s, 1H), 8.34
(t, J=7.6 Hz, 1H), 7.66 (s, 1H), 7.20 (d, J=8.7 Hz, 1H), 7.03 (d,
J=11.8 Hz, 1H), 6.93 (d, J=9.1 Hz, 1H), 6.11-5.77 (m, 3H),
5.33-5.06 (m, 1H), 4.87-4.56 (m, 1H), 4.52-4.14 (m, 1H), 3.97-3.69
(m, 2H), 3.53-3.40 (m, 2H), 3.23-3.11 (m, 1H), 3.11-2.93 (m, 1H),
2.47-2.44 (m, 2H), 2.13-1.96 (m, 2H), 1.68 (d, J=70.9 Hz, 4H), 1.48
(s, 9H), 1.02 (t, J=7.5 Hz, 3H), 0.86-0.68 (m, 2H), -0.17 (s,
9H).
[0202] Preparation 5:
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetr-
ahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid (8')
##STR00022##
[0203] To a stirred solution of
(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1-(t-
etrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilypethoxy)-met-
hyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid
(7') (5.7 g, 7.75 mmol) in 5:1 dioxane:water (60 mL) was added
conc. HCl (20 mL) drop wise at 0.degree. C. The reaction mixture
was warmed and stirred at 90.degree. C. for 16 h and distilled
under vacuum to provide the crude residue, which was sequentially
triturated with chilled diethyl ether and acetonitrile to provide
the HCl salt of the title compound (3.6 g. 95% yield) as a light
brown solid. (m/z): [M+H].sup.+ calcd for
C.sub.22H.sub.20FN.sub.5O.sub.3 422.16 found 422.24. .sup.1H NMR
(400 MHz, D.sub.b 0/DMSO-d.sub.6) .delta. 8.22 (d, J=8.4 Hz, 1H),
7.49 (s, 1H), 7.19 (d, J=8.1 Hz, 1 H), 6.99 (d, J=11.9 Hz, 1 H),
6.91 (d, J =9.0 Hz, 1H), 4.56-4.51 (m, 1H), 4.36 (d, J=15.5 Hz,
1H), 4.30 (d, J=15.5 Hz, 1H), 3.35-3.25 (m, 1H), 3.15-3.05 (m, 1H),
2.4-2.55 (m, 2H), 0.97 (t, J=7.5 Hz, 3H).
[0204] Preparation 6:
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5-
,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid
##STR00023##
[0205] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetr-
ahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, HCl (400 mg,
0.874 mmol) (8') and propionaldehyde (0.095 mL, 1.310 mmol) in DMF
(7 mL), was added sodium cyanoborohydride (165 mg, 2.62 mmol) and
the reaction mixture was stirred at RT overnight. Sodium
borohydride (33 mg, 0.874 mmol) was added, the solution was
concentrated, and purified by preparative HPLC to provide the TFA
salt of the title compound (179 mg, 37% yield). (m/z): [M+H].sup.+
calcd for C.sub.25H.sub.26FN.sub.5O.sub.3 464.20 found 464.5.
[0206] Preparation 7:
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl--
4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6- carboxylic Acid
##STR00024##
[0207] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetr-
ahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, HCl (8') (400
mg, 0.874 mmol), acetone (0.192 mL, 2.62 mmol), and acetic acid
(0.150 mL, 2.62 mmol) in DMF (7 mL), was added sodium
cyanoborohydride (274 mg, 4.37 mmol) and the reaction mixture was
stirred at RT overnight. Sodium borohydride (33 mg, 0.874 mmol) was
added, the solution was concentrated, and purified by preparative
HPLC to provide the TFA salt of the title compound (115 mg, 23%
yield). (m/z): [M+H].sup.+ calcd for
C.sub.25H.sub.26FN.sub.5O.sub.3 464.20 found 464.5.
[0208] Preparation 8:
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4,5-
,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid
##STR00025##
[0209] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetr-
ahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, HCl (8') (300
mg, 0.655 mmol) and 37 wt. % formaldehyde in water (0.059 mL, 0.786
mmol) DMF (5 mL), was added sodium cyanoborohydride (165 mg, 2.62
mmol) and the reaction mixture was stirred at RT overnight. Sodium
borohydride 25 mg, 0.655 mmol) was added, the solution was
concentrated, and purified by flash chromatography (100 g column,
5-75% ACN/water), to provide the TFA salt of the title compound (85
mg, 24% yield). (m/z): [M+H].sup.+ calcd for
C.sub.23H.sub.22FN.sub.5O.sub.3 436.17 found 436.45.
[0210] Preparation 9:
(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,-
6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid
##STR00026##
[0211] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetr-
ahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, HCl (8') (450
mg, 0.983 mmol) and acetaldehyde (0.083 mL, 1.474 mmol) in DMF (7
mL), was added sodium cyanoborohydride (247 mg, 3.93 mmol) and the
reaction mixture was stirred at RT overnight. Sodium borohydride
(112 mg, 2.95 mmol) was added, the solution was concentrated,
dissolved in 1:1 acetic acid: water+300 .mu.L TFA (7 mL) and
purified by flash chromatography (100 g column, 5-65 ACN/water), to
provide the TFA salt of the title compound (165 mg, 0.293 mmol, 30%
yield). (m/z): [M+H].sup.+ calcd for
C.sub.24H.sub.24FN.sub.5O.sub.3 450.19 found 450.
Example 2:
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-h-
ydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo
[4,5-c]pyridin-6-yl)methanone
##STR00027##
[0213] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl--
4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, TFA
(179 mg, 0.310 mmol), N,N-dimethylazetidin-3-amine, 2 HCl (107 mg,
0.465 mmol), and DIPEA (0.162 mL 0.930 mmol) in DMF (4 mL), was
added HATU (177 mg, 0.465 mmol) and the reaction mixture was
stirred at RT overnight. Hydrazine (5 eq) was added, the reaction
mixture was concentrated and purified by preparative HPLC to
provide the TFA salt of the title compound (63 mg, 26% yield).
(m/z): [M+H].sup.+ calcd for C.sub.30H.sub.36FN.sub.7O.sub.2 546.29
found 546.7. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.90 (s,
1H), 8.29 (dd, 1H), 7.34 (s, 1H), 7.07 (d, 1H), 7.01 (d, 1H), 6.89
(d, 1H), 4.35-4.18 (m, 1H), 4.11-3.94 (m, 1H), 3.94-3.73 (m, 3H),
3.70-3.57 (m, 2H), 3.06-2.94 (m, 2H), 2.87-2.66 (m, 2H), 2.48-2.40
(m, 2H), 2.13-2.00 (m, 6H), 1.07 (t, 3H), 1.03-0.93 (m, 6H).
Example 4:
(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-h-
ydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5--
c]pyridin-6-yl)methanone
##STR00028##
[0215] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5-
,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, TFA
(30 mg, 0.052 mmol), N,N-dimethylazetidin-3-amine, 2HCl (27.0 mg,
0.156 mmol), and DIPEA (0.064 mL, 0.364 mmol) in DMF (1.5 mL), was
added HATU (29.6 mg, 0.078 mmol) and the reaction mixture was
stirred at RT overnight. Hydrazine (5 eq) was added, the reaction
mixture was stirred at RT for 10 min, concentrated and purified by
preparative HPLC to provide the TFA salt of the title compound
(29.6 mg, 74% yield). (m/z): [M+H].sup.+ calcd for
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 found 546.6.
Example 8:
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-f-
luoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imi-
dazo[4,5-c]pyridin-6-yl)methanone
##STR00029##
[0217] To a solution of
(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5-
,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid, TFA
(30 mg, 0.052 mmol), N,N-3-trimethylazetidin-3-amine, 2 HCl (29.2
mg, 0.156 mmol), and DIPEA (0.073 mL, 0.416 mmol) in DMF (1 mL),
was added HATU (29.6 mg, 0.078 mmol) and the reaction mixture was
stirred at RT overnight. Hydrazine (5 eq) was added, the reaction
mixture was concentrated and purified by preparative HPLC to
provide the TFA salt of the title compound (24.7 mg, 60% yield).
(m/z): [M+H].sup.+ calcd for C.sub.31H.sub.38FN.sub.7O.sub.2 560.31
found 560.2.
Example 8-22:
(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hyd-
roxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-
imidazo[4,5-c]pyridin-6-yl)methanone
##STR00030##
[0219]
(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl-
)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid,
TFA (30 mg, 0.053 mmol), N,N-dimethylazetidin-3-amine (16 mg, 0.16
mmol), and DIPEA (0.037 mL, 0.213 mmol) were dissolved in DMF (1.0
ml), then HATU (30.4 mg, 0.080 mmol) was added and the reaction
mixture was stirred at room temperature for 6 hours. Hydrazine (15
.mu.L) was added then the solution was concentrated and purified by
preparative HPLC to provide the TFA salt of the title couple (27
mg, 66% yield). (m/z): [M+H]+ calcd for
C.sub.29H.sub.34FN.sub.7O.sub.2 532.6 found 532.2.
Example 8-23:
(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-flu-
oro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-
tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone
##STR00031##
[0221]
(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl-
)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid,
TFA (30 mg, 0.053 mmol), N,N,3-trimethylazetidin-3-amine (18 mg,
0.16 mmol), and DIPEA (0.037 mL, 0.213 mmol) were dissolved in DMF
(1.0 ml), then HATU (30.4 mg, 0.080 mmol) was added and the
reaction mixture was stirred at room temperature for 6 hours.
Hydrazine (15 .mu.L) was added then the solution was concentrated
and purified by preparative HPLC to provide the TFA salt of the
title couple (28 mg, 68% yield). (m/z): [M+H]+ calcd for
C.sub.30H.sub.36FN.sub.7O.sub.2 546.7 found 546.2.
Example 8-14:
(S)-(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5-
,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)(3-(piperidin-1-yl)azetidin--
1-yl)methanone, 2TFA
##STR00032##
[0223]
(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl-
)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid,
TFA (40 mg, 0.071 mmol), 1-(3-azetidinyl)piperidine (29.9 mg, 0.213
mmol), and DIPEA (0.050 ml, 0.284 mmol) were dissolved in DMF (1.5
ml), then HATU (40.5 mg, 0.106 mmol) was added and the reaction
mixture was stirred at room temperature for 2 hours. Hydrazine
(0.011 ml, 0.355 mmol) was added and the reaction mixture was
stirred at room temperature for 10 minutes. The solution was then
concentrated purified by preparative HPLC to provide the TFA salt
of the title compound (36 mg, 63% yield). (m/z): [M+H]+ calcd for
C.sub.32H.sub.38FN.sub.7O.sub.2 572.7 found 572.5.
[0224] Using similar synthetic methods, the compounds of Table 1
were prepared. In the following tables, a blank in any column
indicates a hydrogen atom, a * in a structure heading a table
indicates a chiral center, and the notation (R) or (S) in front of
a substituent denotes the configuration of the carbon atom to which
the substituent is attached.
TABLE-US-00001 TABLE 1 ##STR00033## Ex. Calc Found No. * R.sup.1
R.sup.5 R.sup.6 R.sup.7 Formula [M + H].sup.+ [M + H].sup.+ 8-1
CH.sub.3 --(CH.sub.2).sub.5-- C.sub.31H.sub.36FN.sub.7O.sub.2
558.29 557.7 8-2 CH.sub.3 CH.sub.3 CH.sub.3
C.sub.28H.sub.32FN.sub.7O.sub.2 518.26 517.8 8-3 nPr CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.2 8-4 C.sub.2H.sub.5
--(CH.sub.2).sub.5-- C.sub.32H.sub.38FN.sub.7O.sub.2 572.31 571.7
8-5 nPr --(CH.sub.2).sub.5-- C.sub.33H.sub.40FN.sub.7O.sub.2 586.32
585.7 8-6 iPr --(CH.sub.2).sub.5-- C.sub.33H.sub.40FN.sub.7O.sub.2
586.32 585.9 8-7 iPr CH.sub.3 CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.2 8-8 C.sub.2H.sub.5
CH.sub.3 CH.sub.3 C.sub.29H.sub.34FN.sub.7O.sub.2 532.28 532.2 8-9
CH.sub.3 CH.sub.3 CH.sub.3 CH.sub.3 C.sub.29H.sub.34FN.sub.7O.sub.2
532.28 532.2 8-10 C.sub.2H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.2 8-11 nPr CH.sub.3
CH.sub.3 CH.sub.3 C.sub.31H.sub.38FN.sub.7O.sub.2 560.31 560.2 8-12
iPr CH.sub.3 CH.sub.3 CH.sub.3 C.sub.31H.sub.38FN.sub.7O.sub.2
560.31 560.3 8-13 (R) nPr CH.sub.3 CH.sub.3 CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.5 8-14 (S)
C.sub.2H.sub.5 --(CH.sub.2).sub.5-- C.sub.32H.sub.38FN.sub.7O.sub.2
572.31 572.3 8-15 (R) C.sub.2H.sub.5 --(CH.sub.2).sub.5--
C.sub.32H.sub.38FN.sub.7O.sub.2 572.31 572.2 8-16 (S) iPr CH.sub.3
CH.sub.3 CH.sub.3 C.sub.31H.sub.38FN.sub.7O.sub.2 560.31 560.2 8-17
(R) iPr CH.sub.3 CH.sub.3 C.sub.30H.sub.36FN.sub.7O.sub.2 546.29
546.2 8-18 (R) iPr CH.sub.3 CH.sub.3 CH.sub.3
C.sub.31H.sub.38FN.sub.7O.sub.2 560.31 560.2 8-19 (R) nPr CH.sub.3
CH.sub.3 CH.sub.3 C.sub.31H.sub.38FN.sub.7O.sub.2 560.31 560.6 8-20
(R) C.sub.2H.sub.5 CH.sub.3 CH.sub.3
C.sub.29H.sub.34FN.sub.7O.sub.2 532.28 532.2 8-21 (R)
C.sub.2H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.2 8-22 (S)
C.sub.2H.sub.5 CH.sub.3 CH.sub.3 C.sub.29H.sub.34FN.sub.7O.sub.2
532.28 532.2 8-23 (S) C.sub.2H.sub.5 CH.sub.3 CH.sub.3 CH.sub.3
C.sub.30H.sub.36FN.sub.7O.sub.2 546.29 546.2
Biological Assays
[0225] The compounds of the invention have been characterized in
one or more of the following biological assays.
[0226] Assay 1: Biochemical JAK Kinase Assays
[0227] A panel of four LanthaScreen JAK biochemical assays (JAK1,
2, 3 and Tyk2) were carried in a common kinase reaction buffer (50
mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl.sub.2, and 1 mM EGTA).
Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide
substrate were obtained from Life Technologies.
[0228] Serially diluted compounds were pre-incubated with each of
the four JAK enzymes and the substrate in white 384-well
microplates (Corning) at ambient temperature for 1h. ATP was
subsequently added to initiate the kinase reactions in 10 .mu.L
total volume, with 1% DMSO. The final enzyme concentrations for
JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM
respectively; the corresponding Km ATP concentrations used are 25
.mu.M, 3 .mu.M, 1.6 .mu.M, and 10 .mu.M; while the substrate
concentration is 200 nM for all four assays. Kinase reactions were
allowed to proceed for 1 hour at ambient temperature before a 10
.mu.L preparation of EDTA (10 mM final concentration) and
Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final
concentration) in TR-FRET dilution buffer (Life Technologies) was
added. The plates were allowed to incubate at ambient temperature
for 1h before being read on the EnVision reader (Perkin Elmer).
Emission ratio signals (520 nm/495 nm) were recorded and utilized
to calculate the percent inhibition values based on DMSO and
background controls.
[0229] For dose-response analysis, percent inhibition data were
plotted vs. compound concentrations, and IC.sub.50 values were
determined from a 4-parameter robust fit model with the Prism
software (GraphPad Software). Results were expressed as pIC.sub.50
(negative logarithm of IC.sub.50) and subsequently converted to pKi
(negative logarithm of dissociation constant, Ki) using the
Cheng-Prusoff equation.
[0230] Test compounds having a lower K.sub.i value or higher
pK.sub.i value in each of the four JAK assays show greater
inhibition of JAK activity.
[0231] Assay 2: Cellular JAM Potency Assay
[0232] The AlphaScreen JAM cellular potency assay was carried out
by measuring interleukin-13 (IL-13, R&D Systems) induced STAT6
phosphorylation in BEAS-2B human lung epithelial cells (ATCC). The
anti-STAT6 antibody (Cell Signaling Technologies) was conjugated to
AlphaScreen acceptor beads (Perkin Elmer), while the anti-pSTAT6
(pTyr641) antibody (Cell Signaling Technologies) was biotinylated
using EZ-Link Sulfo-NHS-Biotin (Thermo Scientific).
[0233] BEAS-2B cells were grown at 37.degree. C. in a 5% CO.sub.2
humidified incubator in 50% DMEM/50% F-12 medium (Life
Technologies) supplemented with 10% FBS (Hyclone), 100 U/mL
penicillin, 100 .mu.g/mL streptomycin (Life Technologies), and 2 mM
GlutaMAX (Life Technologies). On day 1 of the assay, cells were
seeded at a 7,500 cells/well density in white poly-D-lysine-coated
384-well plates (Corning) with 254 medium, and were allowed to
adhere overnight in the incubator. On day 2 of the assay, the
medium was removed and replaced with 12 .mu.L of assay buffer
(Hank's Balanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/ml
bovine serum albumin/BSA) containing dose-responses of test
compounds. Compounds were serially diluted in DMSO and then diluted
another 1000-fold in media to bring the final DMSO concentration to
0.1%. Cells were incubated with test compounds at 37.degree. C. for
1 h, and followed by the addition of 12 .mu.l of pre-warmed IL-13
(80 ng/mL in assay buffer) for stimulation. After incubating at
37.degree. C. for 30 min, the assay buffer (containing compound and
IL-13) was removed, and 10 .mu.L of cell lysis buffer (25 mM HEPES,
0.1% SDS, 1% NP-40, 5 mM MgCl.sub.2, 1.3 mM EDTA, 1 mM EGTA, and
supplement with Complete Ultra mini protease inhibitors and
PhosSTOP from Roche Diagnostics). The plates were shaken at ambient
temperature for 30 min before the addition of detection reagents. A
mixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor
beads was added first and incubated at ambient temperature for 2 h,
followed by the addition of streptavidin conjugated donor beads
(Perkin Elmer). After a minimum of 2 h incubation, the assay plates
were read on the EnVision plate reader. AlphaScreen luminescence
signals were recorded and utilized to calculate the percent
inhibition values based on DMSO and background controls. For
dose-response analysis, percent inhibition data were plotted vs.
compound concentrations, and IC.sub.50 values were determined from
a 4-parameter robust fit model with the Prism software. Results may
also be expressed as the negative logarithm of the IC.sub.50 value,
pIC.sub.50.
[0234] Test compounds having a lower IC.sub.50 value or higher
pIC.sub.50 value in this assay show greater inhibition of IL-13
induced STAT6 phosphorylation.
[0235] In Vitro Assay Results
[0236] Selected compounds of the invention were tested in the four
JAK enzyme assays; JAK1, JAK2, JAK3, and Tyk2, and the BEAS-2B
cellular potency assay described above. As shown in Table 19 below,
it was observed that JAK1 enzyme potency was predictive of both
pan-JAK enzyme activity and cellular potency in the BEAS-2B assay.
Therefore, all of the compounds made were tested in the JAK1 enzyme
assay and the BEAS-2B cellular assay and the great majority were
also tested in the JAK3 enzyme assay. All of the compounds
exhibited JAK1 K.sub.i values between 0.04 nM and 0.6 nM (pK.sub.i
between 9.2 and 10.4). The compounds tested in the JAK3 enzyme
assay exhibited K.sub.i values between 0.08 nM and 0.5 nM (pK.sub.i
between 9.3 and 10.1). The compounds tested exhibited IC.sub.50
values in the BEAS-2B assay between 3 nM and 100 nM (pIC.sub.50
between 7 and 8.5).
TABLE-US-00002 TABLE 2 JAK1 JAK2 JAK3 Tyk2 BEAS-2B Example K.sub.i
K.sub.i K.sub.i K.sub.i IC.sub.50 Number (nM) (nM) (nM) (nM) (nM) 2
0.04 0.02 0.10 0.32 4.1 4 0.06 0.13 7.4 8 0.06 0.16 8.5 8-1 0.06
0.18 6.3 8-2 0.06 0.25 12.6 8-3 0.06 0.16 5.0 8-4 0.06 0.13 10.0
8-5 0.08 0.20 25.1 8-6 0.06 0.25 15.8 8-7 0.06 0.20 3.1 8-8 0.08
0.28 5.0 8-9 0.05 0.16 7.1 8-10 0.08 0.32 3.2 8-11 0.10 0.32 6.3
8-12 0.06 0.25 4.0 8-13 0.1 0.32 4.0 8-14 0.08 0.20 12.6 8-15 0.08
0.03 0.40 0.40 5.6 8-16 0.10 0.32 5.0 8-17 0.05 0.13 8.0 8-18 0.10
0.32 4.0 8-19 0.06 0.20 8.0 8-20 0.05 0.20 5.0 8-21 0.05 0.25 4.0
8-22 0.06 0.32 5.25 8-23 0.05 0.2 7.7
[0237] Assay 3: Pharmacokinetics in Plasma and Lung in Mouse
[0238] Plasma and lung levels of test compounds and ratios thereof
were determined in the following manner. BALB/c mice from Charles
River Laboratories were used in the assay. Test compounds were
individually formulated in 20% propylene glycol in pH 4 citrate
buffer at a concentration of 0.2 mg/mL and 50 .mu.L of the dosing
solution was introduced into the trachea of a mouse by oral
aspiration. At various time points (typically 0.167, 2, 6, 24 hr)
post dosing, blood samples were removed via cardiac puncture and
intact lungs were excised from the mice. Blood samples were
centrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at
approximately 12,000 rpm at 4.degree. C. to collect plasma. Lungs
were padded dry, weighed, and homogenized at a dilution of 1:3 in
sterile water. Plasma and lung levels of test compound were
determined by LC-MS analysis against analytical standards
constructed into a standard curve in the test matrix. A lung to
plasma ratio was determined as the ratio of the lung AUC in .mu.g
hr/g to the plasma AUC in .mu.g hr/mL, where AUC is conventionally
defined as the area under the curve of test compound concentration
vs. time. Compounds of the invention exhibited exposure in lung
from one to two orders of magnitude greater than exposure in plasma
in mouse. All of the compounds profiled in this assay exhibited a
half-life between about 4.5 and about 14 hours.
[0239] Assay 4: Murine (Mouse) Model of IL-13 Induced pSTAT6
Induction in Lung Tissue I1-13 is an important cytokine underlying
the pathophysiology of asthma (Kudlacz et al. Eur. J. Pharmacol,
2008, 582,154-161). IL-13 binds to cell surface receptors
activating members of the Janus family of kinases (JAK) which then
phosphorylate STAT6 and subsequently activates further
transcription pathways. In the described model, a dose of IL-13 was
delivered locally into the lungs of mice to induce the
phosphorylation of STAT6 (pSTAT6) which is then measured as the
endpoint.
[0240] Adult balb/c mice from Harlan were used in the assay. On the
day of study, animals were lightly anesthetized with isoflurane and
administered either vehicle or test compound (1 mg/mL, 50 .mu.L
total volume over several breaths) via oral aspiration. Animals
were placed in lateral recumbency post dose and monitored for full
recovery from anesthesia before being returned to their home cage.
Four hours later, animals were once again briefly anesthetized and
challenged with either vehicle or IL-13 (0.03 .mu.g total dose
delivered, 50 .mu.L total volume) via oral aspiration before being
monitored for recovery from anesthesia and returned to their home
cage. One hour after vehicle or IL-13 administration, lungs were
collected for both pSTAT6 detection using an anti-pSTAT6 ELISA
(rabbit mAb capture/coating antibody; mouse mAb detection/report
antibody: anti-pSTAT6-pY641; secondary antibody: anti-mouse
IgG-HRP) and analyzed for total drug concentration as described
above in Assay 3.
[0241] Selected compounds of the invention were tested in the
assay. Activity in the model is evidenced by a decrease in the
level of pSTAT6 present in the lungs of treated animals at 5 hours
compared to the vehicle treated, IL-13 challenged control animals.
The difference between the control animals which were vehicle-
treated, IL-13 challenged and and the control animals which were
vehicle-treated, vehicle challenged dictated the 0% and 100%
inhibitory effect, respectively, in any given experiment. Exemplary
compounds of the invention were tested in the assay, and exhibited
inhibition of STAT6 phosphorylation at 4 hours after IL-13
challenge as documented below.
[0242] Confirming the relevance of the JAK-STAT pathway in airway
inflammation, compounds which have demonstrated in vivo target
engagement in the IL13-induced pSTAT6 mouse model are subsequently
tested and proven to be efficacious in a mouse model of
allergen-induced eosinophilic inflammation.
In Vivo Assay Results
[0243] Selected compounds of the invention were characterized in
both the pharmacokinetic assay (Assay 3) and pharmacodynamic assay
(Assay 4). A good correlation was observed between test compound
concentration in lung determined in the pharmacokinetic assay and
in the pharmacodynamic assay at a similar time points post dosing.
Observation of significant compound concentration in the mouse lung
in the pharmacodynamic assay confirmed that the observed inhibition
of IL-13 induced pSTAT6 induction was a result of the activity of
the test compound.
[0244] In the following table, for the ratio of lung exposure to
plasma exposure (Assay 3), A denotes a ratio 100-200, B denotes a
ratio between 50 and 100, and C denotes a ratio between 20 and 50.
For the per cent inhibition of IL-13 induced pSTAT6 induction
(Assay 4), A represents between 60% and 80% inhibition, B
represents between 40% and 60% inhibition and C represents between
25% and 40% inhibition.
TABLE-US-00003 TABLE 3 Lung to Plasma pSTAT6 Example ratio
inhibition Number Assay 3 Assay 4 2 A B 4 C A 8 B B 8-3 C B 8-7 C B
8-13 B 8-15 C 8-16 B C 8-22 B B 8-23 B B
[0245] Assay 5: Murine Model of Alternaria Alternata-Induced
Eosinophilic Inflammation of the Lung
[0246] Airway eosinophilia is a hallmark of human asthma.
Alternaria alternata is a fungal aeroallergen that can exacerbate
asthma in humans and induces eosinophilic inflammation in the lungs
of mice (Havaux et al. Clin Exp Immunol. 2005
February;139(2):179-88). In mice, it has been demonstrated that
alternaria indirectly activates tissue resident type 2 innate
lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)
and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and
coordinate eosinophilic inflammation (Bartemes et al. J Immunol.
2012 Feb. 1; 188(3):1503-13).
[0247] Seven- to nine-week old male C57 mice from Taconic were used
in the study. On the day of study, animals were lightly
anesthetized with isoflurane and administered either vehicle or
test compound (0.03-1.0 mg/mL, 50 .mu.L total volume over several
breaths) via oropharyngeal aspiration. Animals were placed in
lateral recumbency post dose and monitored for full recovery from
anesthesia before being returned to their home cage. One hour
later, animals were once again briefly anesthetized and challenged
with either vehicle or alternaria extract (200 ug total extract
delivered, 50 .mu.L total volume) via oropharyngeal aspiration
before being monitored for recovery from anesthesia and returned to
their home cage. Forty-eight hours after alternaria administration,
bronchoalveolar lavage fluid (BALF) was collected and eosinophils
were counted in the BALF using the Advia 120 Hematology System
(Siemens).
[0248] Selected compounds of the invention demonstrating in vivo
activity in the IL-13-pSTAT6 pharmacodynamic assay were tested in
this alternaria assay. Activity in the model is evidenced by a
decrease in the level of eosinophils present in the BALF of treated
animals at forty-eight hours compared to the vehicle treated,
alternaria challenged control animals. Data are expressed as
percent inhibition of the vehicle treated, alternaria challenged
BALF eosinophils response. To calculate percent inhibition, the
number of BALF eosinophils for each condition is converted to
percent of the average vehicle treated, alternaria challenged BALF
eosinophils and subtracted from one-hundred percent. Exemplary
compounds of the invention were tested in the assay and exhibited
inhibition of BALF eosinophil counts at forty-eight hours after
alternaria challenge as documented below.
In Vivo Assay Results
[0249] All of the compounds tested demonstrated a range of
inhibition (73%-93%) of alternaria-induced BALF eosinophils. The
following table reflects the maximum statistically significant
percent inhibition of the vehicle treated, alternaria challenged
level of eosinophil induction.
TABLE-US-00004 TABLE 4 Percent Inhibition of Example
Alternaria-induced Number BALF Eosinophils 2 74 4 79
[0250] Assay 6: IL-5 Mediated Eosinophil Survival Assay
[0251] The potency of the test compound for IL-5 mediated
eosinophil survival was measured in human eosinophils isolated from
human whole blood (AllCells). Because IL-5 signals through JAK,
this assay provides a measure of JAK cellular potency.
[0252] Human eosinophils were isolated from fresh human whole blood
(AllCells) of healthy donors. Blood was mixed with 4.5% Dextran
(Sigma-Aldrich) in a 0.9% sodium chloride solution (Sigma-Aldrich).
Red blood cells were left to sediment for 35 minutes. The leukocyte
rich upper layer was removed and layered over Ficoll-Paque (GE
Healthcare) and centrifuged at 600 g for 30 minutes. The plasma and
mononuclear cell layers were removed before the granulocyte layer
was lysed with water to remove any contaminating red blood cells.
Eosinophils were further purified using a human eosinophil
isolation kit (Miltenyi Biotec). A fraction of the purified
eosinophils were incubated with anti-CD16 FITC (Miltenyi Biotec)
for 10 minutes at 4.degree. C. in the dark. Purity was analyzed
using a LSRII flow cytometer (BD Biosciences).
[0253] Cells were cultured in a 37.degree. C., 5% CO.sub.2
humidified incubator in RPMI 1640 (Life Technologies) supplemented
with 10% Heat Inactivated Fetal Bovine Serum (FBS, Life
Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life
Technologies) and 1X Pen/Strep (Life Technologies). Cells were
seeded at 10,000 cells/well in media (50 .mu.L). The plate was
centrifuged at 300 g for 5 minutes and supernatants removed.
Compounds were serially diluted in DMSO and then diluted another
500-fold to a 2.times. final assay concentration in media. Test
compounds (50 .mu.L/ well) were added to cells, and incubated at
37.degree. C., 5% CO.sub.2 for 1 hour, followed by the addition of
IL-5 (R&D Systems; final concentrations 1 ng/mL and 10 pg/ml)
in pre-warmed assay media (50 .mu.L) for 72 hours.
[0254] After cytokine stimulation, cells were centrifuged at 300 g
for 5 min and washed twice with cold DPBS (Life Technologies). To
access viability and apoptosis, cells were incubated with Propidium
Iodide (Thermo Fisher Scientific) and APC Annexin V (BD
Biosciences) and analyzed using a LSRII flow cytometer (BD
Biosciences). IC.sub.50 values were determined from analysis of the
viability curves of percent cell viability vs compound
concentration. Data are expressed as pIC.sub.50 (negative decadic
logarithm IC.sub.50 ) values. The compound of example 2 exhibited a
pIC.sub.50 value of 7.6.+-.0.5 in the presence of 10 pg/ml IL-5 and
a pIC.sub.50 value of 6.2.+-.0.1 in the presence of 1 ng/ml
IL-5.
[0255] Assay 7: Inhibition of IFN.gamma. and IL-27 induced
chemokines CXCL9 and CXCL10 in human 3D airway cultures
[0256] EpiAirway tissue cultures were obtained from Mattek
(AIR-100). Cultures were derived from asthmatic donors. In a cell
culture insert, human derived tracheal/bronchial epithelial cells
were grown and differentiated on a porous membrane support,
allowing an air-liquid interface with warmed culture medium below
the cells and a gaseous test atmosphere above. Tissues were
cultured in maintenance media (Mattek, AIR-100-MM) in a 37.degree.
C., 5% CO2 humidified incubator. Four donors were tested. On Day 0,
tissue cultures were treated with test compounds at 10 .mu.M, 1
.mu.M and/or 0.1 .mu.M. Compounds were diluted in dimethyl
sulfoxide (DMSO, Sigma) to a final concentration of 0.1%. DMSO at
0.1% was used as a vehicle control. Test compounds were incubated
with cultures for 1 hour at 37.degree. C., 5% CO.sub.2, followed by
the addition of pre-warmed media containing IFN.gamma. (R&D
Systems) or IL-27 (R&D Systems) at a final concentration at 100
ng/ml. Tissue cultures were maintained for 8 days. Media was
replaced every 2 days with fresh media containing compounds and
IFN.gamma. or IL-27. On Day 8, tissue cultures and supernatants
were collected for analysis. Supernatant samples were assayed for
CXCL10 (IP-10) and CXCL9 (MIG) using luminex analysis (EMD
Millipore). Data is expressed as % Inhibition +/-standard deviation
(.+-.STDV). Percent inhibition was determined by compound
inhibitory potency against IFN.gamma. or IL-27 induced CXCL10 or
CXCL9 secretion compared to vehicle treated cells. Data is the
average from 3 or 4 donors. The compound of example 2 was able to
inhibit IFN.gamma. induced CXCL10 secretion by 101%.+-.2.0 (at 10
.mu.M), 65% .+-.29 (at .mu.M) and 6%.+-.11 (at 0.1 .mu.M) when
compared to vehicle control. The compound of example 2 was able to
inhibit IFN.gamma. induced CXCL9 secretion by 93%.+-.13 (at 10
.mu.M) and 24%.+-.49 (at 1 .mu.M) when compared to vehicle. The
compound of example 2 was able to inhibit IL-27 induced CXCL10
secretion by 108% .+-.11 (at 10 .mu.M), 101%.+-.6 (at 1 .mu.M) and
69%.+-.10 (at 0.1 .mu.M) when compared to vehicle control. The
compound of example 2 was able to inhibit IL-27 induced CXCL9
secretion by 100%.+-.0 (at 10 .mu.M), 97%.+-.3.6 (at 1 .mu.M) and
57%.+-.28 (at 0.1 .mu.M) when compared to vehicle control.
[0257] Assay 8: Cellular JAK Potency Assay: Inhibition of
IL-2/Anti-CD3 Stimulated IFN.gamma. in Human PBMCs
[0258] The potency of the test compound for inhibition of
interleukin-2 (IL-2)/anti-CD3 stimulated interferon gamma
(IFN.gamma.) was measured in human peripheral blood mononuclear
cells (PBMCs) isolated from human whole blood (Stanford Blood
Center). Because IL-2 signals through JAK, this assay provides a
measure of JAK cellular potency.
[0259] (1) Human peripheral blood mononuclear cells (PBMC) were
isolated from human whole blood of healthy donors using a ficoll
gradient. Cells were cultured in a 37.degree. C., 5% CO.sub.2
humidified incubator in RPMI (Life Technologies) supplemented with
10% Heat Inactivated Fetal Bovine Serum (FBS, Life Technologies), 2
mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies)
and 1X Pen/Strep (Life
[0260] Technologies). Cells were seeded at 200,000 cells/well in
media (50 .mu.L) and cultured for 1 h. Compounds were serially
diluted in DMSO and then diluted another 500-fold (to a 2.times.
final assay concentration) in media. Test compounds (100
.mu.L/well) were added to cells, and incubated at 37.degree. C., 5%
CO.sub.2 for 1 h, followed by the addition of IL-2 (R&D
Systems; final concentration 100 ng/mL) and anti-CD3 (BD
Biosciences; final concentration 1 .mu.g/mL) in pre-warmed assay
media (50 .mu.L) for 24 h.
[0261] (2) After cytokine stimulation, cells were centrifuged at
500 g for 5 min and supernatants removed and frozen at -80.degree.
C. To determine the inhibitory potency of the test compound in
response to IL-.sup.2/anti-CD3, supernatant IFN.gamma.
concentrations were measured via ELISA (R&D Systems). IC.sub.50
values were determined from analysis of the inhibition curves of
concentration of IFN.gamma. vs compound concentration. Data are
expressed as pIC.sub.50 (negative decadic logarithm IC.sub.50)
values. The compound of Example 2 exhibited a pIC.sub.50 value of
about 7.1 in this assay.
[0262] Assay 9: Cellular JAK Potency Assay: Inhibition of IL-2
Stimulated pSTAT5 in CD4+ T Cells
[0263] The potency of the test compound for inhibition of
interleukin-2 (IL-2)/anti-CD3 stimulated STATS phosphorylation was
measured in CD4-positive (CD4+) T cells in human peripheral blood
mononuclear cells (PBMCs) isolated from human whole blood (Stanford
Blood Center) using flow cytometry. Because IL-2 signals through
JAK, this assay provides a measure of JAK cellular potency.
[0264] CD4+ T cells were identified using a phycoerythrobilin (PE)
conjugated anti-CD4 antibody (Clone RPA-T4, BD Biosciences), while
an Alexa Fluor 647 conjugated anti-pSTAT5 antibody (pY694, Clone
47, BD Biosciences) was used to detect STATS phosphorylation.
[0265] (1) The protocol of Assay 8 paragraph (1) was followed with
the exception that the cytokine stimulation with anti-CD3 was
performed for 30 min instead of 24 h.
[0266] (2) After cytokine stimulation, cells were fixed with pre
warmed fix solution (200 .mu.L; BD Biosciences) for 10 min at
37.degree. C., 5% CO.sub.2, washed twice with DPBS buffer (1 mL,
Life Technologies), and resuspended in ice cold Perm Buffer III
(1000 .mu.L, BD Biosciences) for 30 min at 4.degree. C. Cells were
washed twice with 2% FBS in DPBS (FACS buffer), and then
resuspended in FACS buffer (100 .mu.L) containing anti-CD4 PE (1:50
dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for
60 min at room temperature in the dark. After incubation, cells
were washed twice in FACS buffer before being analyzed using a
LSRII flow cytometer (BD Biosciences). To determine the inhibitory
potency of test compounds in response to IL-2/anti-CD3, the median
fluorescent intensity (MFI) of pSTAT5 was measured in CD4+ T cells.
IC.sub.50 values were determined from analysis of the inhibition
curves of MFI vs compound concentration. Data are expressed as
pIC.sub.50 (negative decadic logarithm IC.sub.50) values. The
compound of Example 2 exhibited a pIC.sub.50 value of about 7.3 in
this assay.
[0267] Assay 10: Cellular JAK Potency Assay: Inhibition of IL-4
Stimulated pSTAT6 in CD3+ T cells
[0268] The potency of the test compound for inhibition of
interleukin-4 (IL-4) stimulated STAT6 phosphorylation was measured
in CD3-positive (CD3+) T cells in human peripheral blood
mononuclear cells (PBMCs) isolated from human whole blood (Stanford
Blood Center) using flow cytometry. Because IL-4 signals through
JAK, this assay provides a measure of JAK cellular potency.
[0269] CD3+ T cells were identified using a phycoerythrobilin (PE)
conjugated anti-CD3 antibody (Clone UCHT1, BD Biosciences), while
an Alexa Fluor 647 conjugated anti-pSTAT6 antibody (pY641, Clone
18/P, BD Biosciences) was used to detect STAT6 phosphorylation.
[0270] Human peripheral blood mononuclear cells (PBMC) were
isolated from human whole blood of healthy donors as in Assays 8
and 9. Cells were seeded at 250,000 cells/well in media (200
.mu.L), cultured for 1 h and then resuspended in assay media (50
.mu.L) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2
mM Glutamax, 25 mM HEPES and lx Penstrep) containing various
concentrations of test compounds. Compounds were serially diluted
in DMSO and then diluted another 500-fold (to a 2.times. final
assay concentration) in assay media. Test compounds (50 .mu.L) were
incubated with cells at 37.degree. C., 5% CO.sub.2 for 1 h,
followed by the addition of IL-4 (50 .mu.L) (R&D Systems; final
concentration 20 ng/mL) in pre-warmed assay media for 30 min. After
cytokine stimulation, cells were fixed with pre-warmed fix solution
(100 .mu.L) (BD Biosciences) for 10 min at 37.degree. C., 5%
CO.sub.2, washed twice with FACS buffer (1 mL) (2% FBS in DPBS),
and resuspended in ice cold Perm Buffer III (1000 .mu.L) (BD
Biosciences) for 30 min at 4.degree. C. Cells were washed twice
with FACS buffer, and then resuspended in FACS buffer (100 .mu.L)
containing anti-CD3 PE (1:50 dilution) and anti-pSTAT6 Alexa Fluor
647 (1:5 dilution) for 60 min at room temperature in the dark.
After incubation, cells were washed twice in FACS buffer before
being analyzed using a LSRII flow cytometer (BD Biosciences).
[0271] To determine the inhibitory potency of the test compound in
response to IL-4, the median fluorescent intensity (MFI) of pSTAT6
was measured in CD3+ T cells. IC.sub.50 values were determined from
analysis of the inhibition curves of MFI vs compound concentration.
Data are expressed as pIC.sub.50 (negative decadic logarithm
IC.sub.50 ). The compound of Example 2 exhibited a pIC.sub.50 value
of 7.9 in this assay.
[0272] Assay 11: Cellular JAK Potency Assay: Inhibition of IL-6
Stimulated pSTAT3 in CD3+ T Cells
[0273] A protocol analogous to that of Assay 10 was used to
determine the potency of the test compound for inhibition of
interleuken-6 (IL-6) stimulated STAT3 phosphorylation. An Alexa
Fluor 647 conjugated anti-pSTAT3 antibody (pY705, Clone 4/P, BD
Biosciences) was used to detect STAT3 phosphorylation.
[0274] The compound of Example 2 exhibited a pIC.sub.50 value of
7.2 in this assay.
[0275] Crystal Structure
[0276] A co-crystal structure was obtained of the compound of
Example 2 bound to human JAK1 at a resolution of 2.28 .ANG.. The
ligand was observed to bind in the ATP binding site. Seven specific
hydrogen bonding interactions were identified based upon a distance
of 3.5 .ANG. or less between donor and acceptor atoms. Of
particular note, a hydrogen bonding interaction was identified
between the carbonyl of the exocyclic amide of the compound of
Example 2 and the sidechain of Arg879 of JAK1. In earlier modeling
studies this interaction had been proposed as a way to provide
selectivity for JAK1 over other tyrosine kinases, as otherwise
closely related kinases (e.g. TRKA, VEGFR, ABL1) did not possess an
arginine residue at the equivalent location. The observed results
of the hydrogen bonding interaction in the crystal structure and
improved kinome selectivity compared to series not possessing the
exocyclic amide validate this design hypothesis.
[0277] While the present invention has been described with
reference to specific aspects or embodiments thereof, it will be
understood by those of ordinary skilled in the art that various
changes can be made or equivalents can be substituted without
departing from the true spirit and scope of the invention.
Additionally, to the extent permitted by applicable patent statutes
and regulations, all publications, patents and patent applications
cited herein are hereby incorporated by reference in their entirety
to the same extent as if each document had been individually
incorporated by reference herein.
* * * * *